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1 - Research Programs Unit

1. cceeeeeeseeesseeeeseeeeseeseeeeeeeees 43 Cleaning the Sample Line ccccssseecesseeeeneeeeeseeseesenens 45 Automatic Calibration cccccsssceesseeeeseesesseeeeeneesenseseenes 46 Creating an Experiment ccccsescesesseseeseeeeseeseeaseseeseesens 47 Creating a New Experiment eesssssssoserssssssssseeerersssssssseeresees 47 PCOUMING D ta sna 49 Acquisition Tab Main Functions sssseeeesssessesoeenssssssssseeresees 50 Controlling Data Acquisition and Recording eee 50 Customizing the Worksheet ccccccccssessseeeeeeeeeeeeeeseseeeeeeees 50 Analyzing Data jose cc oesewcscanceuccetcswcteeuecssacnpeiesseeosmedendensvetensnecs 51 SONIN sisii a ia 53 Two way lube SOrine cenoria nn neta Geaieeiiaentel aot 53 Sorting onto 8 well Strips SH800 and SH8002 0 55 Sorting into Multi well Plates SH800ZP only eee 58 Performing Fluorescence Compensation 0008 61 Flushing the Fluidics System ccccseseeeeseeeeeeeeeeeneeeeneees 64 SHUTAOWIT wcsiicisecicsencteveeiwntentnst wetdveaetvetehwendicncdvccumneuseteentenctvcess 65 Specifying User Preferences cscccssseeseseeeenseeeenseeees 66 Exchanging the Sorting Chip ccsssscessssseeceeseeeseenseees 67 Backing Up Restoring the Database ssssssseeeees 68 Backine Up the Database secrncctstasineet neptsan ta a a 68 ReStOriie alata DAS E hape
2. i a 2 a i BEN EE E EH a Ee eS EN i m BE JUE 100 100 100 100 100 100 a 100 100 ca 100 After one set has been sorted the following message appears Sorting SH800 Software A Sorting is completed for all well Do you want to continue to next sort 10 Click Finish to finish sorting To continue sorting click Continue When you click Continue the collection stage is unloaded allowing you to place the next set Performing Fluorescence Compensation Fluorescence compensation is used to remove fluorescence spillover components from each fluorescence detector to obtain the signal for the target fluorochrome only for each detector In fluorescence compensation it is necessary to record a compensation panel comprising an unstained negative control and up to six singly stained positive control samples for each fluorochrome This section describes the fluorescence compensation procedure using the Compensation Wizard For details about using a gate for recording a control tube or adjusting fluorescence compensation manually see Adjusting Fluorescence Compensation Manually page 81 Do not change the gain of the channel detectors while recording the control tubes or samples for a compensation panel The fluorescence compensation process is valid only for the instrument settings in place when all control tubes in a compensation
3. sishjeuy g Je deyug 84 Editing Plots SH800 Software displays data on the following types of plots You can change the plot type change the axis scale and modify other aspects of plots For details about the buttons on the Plot Tools tab of the ribbon see Plot Tools Tab ribbon page 129 For details about adding gates to a plot see Adding Gates page 88 Density plots Density plots display a frequency distribution of fluorescence intensity for two acquisition parameters bivariate distribution Density plots use color to show the distribution of the number of detected events having the same value High density regions are shown in red low density regions are shown in violet and regions of intermediate density are shown using the colors of the visible spectrum in between Density plots are most useful for displaying populations where the number of detected events is relatively large All Events 1 000 800 600 CAA 400 200 a25 125 1 000 Dot plots Dot plots display a frequency distribution of fluorescence intensity for two acquisition parameters bivariate distribution Dot plots are most useful for displaying populations where the number of detected events is relatively small Events on a dot plot are colored based on their classification gate and population Tip As the number of events on a dot plot increases the plot may become saturated
4. Fluorescent lights passes through an array of longpass optical filters LPFs separating the fluorescent light into individual fluorescent channels Each color then passes through a bandpass optical filter and is detected and amplified by a PMT detector Incident light Shorter PF Longer Shorter PF Longer Shorter PF Longer Shorter Pr Longer Shorter Pr Longer FL The SH800 can detect six channels of fluorescent light simultaneously The laser configuration and the optical filter pattern determine the fluorescent markers that can be detected For a list of fluorochromes that can be detected for each model see Fluorochrome Detection Matrix page 204 The output from the photodiode and PMT detectors is passed to the acquisition module The pulse shape that is detected by each detector varies depending on the size and type of cell and the type of light being collected The SH800 measures three parameters per pulse area height and width to provide a wealth of information about cells The width of the pulse provides an indication of the size of a cell the height of the pulse provides an indication of the peak fluorescence intensity brightness of the cell and the area provides an indication of the total fluorescent material contained in the cell The pulse parameters can be selected independently for each channel Intensity Area Height Time q Width When sorting or analyzing cells you can
5. Open Data Source Displays the data source on the worksheet Delete Deletes the selected data source Rename Renames the selected sample tube Export FCS File Displays the FCS File Export dialog page 117 for exporting the selected data source in FCS 3 0 or FCS 3 1 format Show Results Displays the Data Source Results dialog page 115 for displaying the recording results and sorting results for the selected data source Worksheet Context Menu This section describes the context menu items displayed for items displayed on the worksheet and the worksheet itself Worksheet The following menu commands are displayed when right clicking on the worksheet Refresh Updates the information displayed on the worksheet New Density Adds a new density plot to the worksheet New Dot Plot Adds a new dot plot to the worksheet New Histogram Adds a new histogram plot to the worksheet Show Table Shows hides the Gates and Statistics table Show Grid Displays the grid on the worksheet Snap to Grid Aligns the plots and Gates and Statistics table on the worksheet with the grid when they are moved or added Auto Arrange Automatically aligns the plots and Gates and Statistics table on the worksheet to the grid Fit to Screen Expands the worksheet to fill the entire screen Edit Gate Displays the Gate Editor dialog page 127 to edit gates Edit Statistics Displays the Statis
6. The laser initialization timed out Check that the compressed air supply is supplying rated pressure Shut down the cytometer and turn off the compressed air supply Clean or replace the sample loader O ring If the problem persists contact your Sony distributor for service Refill the sheath tank See Refilling the Sheath Tank page 146 Replace the waste tank See Emptying Changing the Waste Tank page 148 Refill the ethanol tank See Refilling the Ethanol Tank page 151 Check that the compressed air supply is supplying rated pressure Connect or reconnect the cart connection cable between the main unit and the fluidics cart The cytometer may be overheating Shut down the cytometer and allow the temperature to fall within the rated range before rebooting If the problem persists at temperatures not exceeding 35 C 95 F contact your Sony distributor for service Shut down the cytometer and wait a while before trying again If the problem persists contact your Sony distributor for service Open the fluidics maintenance door and check for leakages If the problem persists contact your Sony distributor for service Check that the sheath tank is full and is positioned correctly in the fluidics cart If the problem persists contact your Sony distributor for service Check that the waste tank is empty and is positioned correctly in the fluidics cart If the problem persists
7. Tube Information Worksheet Settings Stop amp Sort Settings 2g 2 Way Data Source 1 4 Tube 2 1 amp Tube Information Worksheet Settings Stop amp Sort Settings Bet 2 Way Data Source 1 E3 Experiment 12 28 2013 10 27 57 PM EEJ Experiment 12 2 E Experiment 12 2 EE Experiment 12 28 2013 9 45 52 PM EE Experiment 12 26 2013 8 03 31 PM EE Experiment 12 2 EE 12 26 2013 4 12 46 PM EE Experiment 12 26 2013 3 58 20 PM E Experiment 12 26 2013 3 42 31 PM EE Experiment 12 26 2013 1 19 41 PM EE Experiment 12 2 EE Experiment 12 25 2013 1 46 23 PM EE Experiment 12 24 2013 7 14 49 PM 8 2013 10 26 27 PM 8 2013 10 13 35 PM 6 2013 4 26 01 PM 5 2013 7 22 50 PM Acquisition Experiments 2 Search criteria You can search experiments by date and keyword The search results are displayed in the Experiment Explorer For details about searching see Searching Experiments page 77 Experiment Explorer Displays a list of all current experiments The internal structure of each experiment is displayed in a hierarchical list view public Lists shared experiments Shared experiments can be viewed by all users User_name Lists private experiments created by the logged in user Private experiments cannot be viewed by other users An experiment contains the following structural components Experiment Experiment Information Contains information about the
8. Adjusting the Sort Position SH800ZP only You can adjust the position where droplets fall within wells when sorting into a multi well plate The description in this section shows a 96 well plate as an example The operation is the same for all other multi well plates When sorting into a 384 well plate or 384 well PCR plate always adjust the sort position before starting Tip The sort position cannot be adjusted when running 2 way tube sorting Place a cover over the multi well plate so that you can see the position where droplets fall 2 Select a multi well plate sorting method in Sorting Method in the Sort Control pane and click Sort Settings wt Sort Control B ie i pi Load Collection Sortmg Method 96 Well Plate The Sort Settings dialog appears 3 Adjust the sort position on the Plate Adjustment tab Adjust the position where droplets fall by visually monitoring the top of the multi well plate Adjusting the Sort Position SH800ZP only 99 uos 9g Jsaydeuy 100 2 Move the position mm to the right using gt and 1 5 mm down using Y in Droplet Position 3 Click Start in Sort Test 4 Click Unload 5 Visually check the position of the droplets on the multi well plate cover 6 Repeat the procedure for wells A12 and H12 as required Plate Sort Settings Plate A Please select the target wel Select Target Well To set the adjusted positio
9. Apply Template dialog The Apply Template dialog is displayed by selecting a new sample group or sample tube in the Experiment Explorer and clicking Apply Template on the Experiment tab of the ribbon The Apply Template dialog is used to select a template to apply to the sample group or sample tube SampleGroup Templates Public Templates erCP Cy5 5 My Templates Recent Sample Groups lt 8 _ Cees The configurable items are the same as the Add Sample Group dialog page 112 or Add Tube dialog page 113 for sample groups Tube Results dialog The Tube Results dialog is displayed by selecting a sample tube in the Experiment Explorer and clicking Show Results in the Settings and Information group on the Experiment tab of the ribbon The Tube Results dialog is used to check the recording results and sorting results of the selected sample tube e Basic Information tab Displays basic information identifying the sample tube Chip ID Displays the ID number of the sorting chip Nozzle Size Displays the nozzle size of the sorting chip Main Window uondos q mopuiM Z JeIdeyD I 115 uondosaqg mopuiM Z Je deuyD 116 e Recording Result tab Displays recording results Tube 6 Tube Results Bsc 30 0 FL4 40 0 4 FLi 40 0 FL2 40 0 4 4 FL6 40 0 Record Count Displays the number of events recorded Start Time Displays the time when recording st
10. Checking Device Connections cccceeseeeeeeseseeseesenneeees 29 Filling the DI Water Tank cccssccsesseeeseeceneeeeseeeesseenseeenes 30 Filling the Sheath Fluid Tank cccssscccsssecseseeseeeeseees 30 Filling the Ethanol Tank ccccesscseseeeeseeeeseeeeseeeaseeeneeeoaes 31 Preparing a Sample sisikira aaaeei 31 Loading a Sample Tube in a Sample Tube Holder 0 00 31 Preparing Automatic Setup Beads ccseeseseeeceeeeeeeeeenees 32 Sample Temperature Control sanieirernante iarna 32 Preparing Collection Tubes s ssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 33 Loading 8 well Strips SH800 and SH8002 0 eee 35 Attaching the Splash Guard for use with multi well plates COPIOOOZ P ONI iea a Mevaeedeieas 35 Loading a Multi well Plate SH800ZP only eee 36 Loading a 384 well PCR Plate sc scccssssstesiacsicaeasdbewrudactonrteaeesad 36 Removing the Plate Holder Adapter SH800ZP only 37 Configuring USCIS rannira aaan raa aii 38 Adding USCTS ee Siesta 38 Editing User SeN S arerin cerisraierneet isnt 38 Changing Passwords ccasccesuse sales demnarnisesaveaseqraresuslayalesdecnameiseaaes 39 Chapter 3 Basic Operation WV OF KTIOW eos cclcoetiastcesdenctenteestecetsed sene sesteeuanwiudiewctunteareesseeeoaest 40 System SAUD esccs sees csseecsescctesacssssss vandencevaseneauneecacies seuss 41 LOgONO Mers a adencsersendeavecaaneentacesie 42 Initial Instrument Setup
11. If the problem persists replace the sorting chip If the problem persists contact your Sony distributor for service Chip does not eject Use the manual load unload thumbwheel on the chip loader to remove the chip Troubleshooting 215 snosuel aosi g xlpueddy 216 Possible Cause Recommended Solution Sorting chip clogs after changing the There may be some small particles in the replacement filter that have not been sheath filter removed Run the sheath filter de bubble function at least three times Sample tube hits the sample probe Use only standard tubes and fully insert the tube in the sample tube holder when loading a sample An ill fitting or non standard tube can damage the sample probe Fluid is leaking from the sample probe Check that the sample line is fully inserted in the pinch valve Fluid is leaking from the top in the sorting The sample fluid connector may not be securely fastened Securely fasten the area sample fluid connector Fluidics component pump fluid The seals may have deteriorated due to age causing ethanol or waste fluid to connectors etc is leaking fluid leak Clean the area and contact your Sony distributor for service Always wear gloves and other protective clothing mask and goggles as required when cleaning spills and leakages Droplet camera image is erratic The droplet camera may require cleaning Contact your Sony distributor for service Droplets are not being f
12. Print Prints analysis results and other reports Database Displays commands used to perform experiment database operations and to check hard disk free capacity Help Displays commands used to display the Operator s Guide and to check software version information Log Displays commands used to display the chip alignment log sorting calibration log and user log Restart Restarts SH800 Software When the software restarts it loads the same conditions that existed at the time Restart was pressed but without assigning any experiment as the active experiment Tip The restart function should only be used when the SH800 Software is running very slowly or is otherwise unstable It should not be used during normal operation Logout Logs out the currently logged in user and displays the login window Exit Exits SH800 Software Operation area Displays function buttons and information related to the menu item selected on the left side SH800 Software Operating Environment The host computer must satisfy the following requirements in order to run SH800 Software Operating system OS Microsoft Windows 8 Operating System Professional 64 bit CPU Intel Core i17 3820QM 2 70 GHz or later Cache Level 3 cache memory 8 MB or greater Memory PC3 12800 1600 MHz 16 GB 8 GB x 2 or greater Network interface 1000BASE T 1 Main storage 1 TB HDD SATA 1 TB x 1 or greater Monitor resolution 1920
13. Carefull cleaning 30ml centrifuge tube with 30ml of sodium O Keep DI water inside sample probe DI water cleaning starts When cleaning with DI water finishes the following screen appears Shutdown 65 uoneiedo oiseg g13 deyo Kii uoleisdo olseg sa deuyD 66 Click OK to shut down the SH800 Shutdown Wizard 3 Shutdown Do you want to shut down The fluidics system is depressurized and all fluidics lines are sealed Specifying User Preferences Specifying User Preferences Various configuration settings and values can be saved by each user in user preferences User preferences are loaded automatically each time a user logs in saving having to reconfigure the instrument each time you want to acquire or analyze data 1 Click Information on the File tab of the ribbon then click User Preference ia Change Password rent password Change your cu 2 Institution Information v your institution information Editable by administrator only g User Preference Change software default settings for each use The User Preference dialog appears 2 Set each item The values set here become the default values for each setting User Preference Cytometer Define default settings for sort control Reset Sort Settings Experiment Define default sorting method Worsted A Sorting method Compensation 2 Way Tubes User Defined Color Plate Position FCS File
14. Editing Plots page 84 Editing Gates You can change the shape of and settings for gates on plots To change the shape of a gate Select the gate and drag the drawing handles to change the shape For quadrant gates drag the intersection point of the quadrants to another position to change the gate To move a gate Select the gate and drag the gate to the desired position To rotate a gate You can rotate polygon and ellipse gates Select a polygon or ellipse gate and drag the rotation handle to rotate the gate Rotation handle All Events All Events To change gate settings Select the gate click Edit Gate in the Gate group on the Plot Tools tab of the ribbon to display the Gate Editor dialog and then select the Details tab FITC Gate Editor Remove Gate Properties For details about each item see Gate Editor dialog page 127 To delete a gate Select the gate to be deleted then click Remove Gate in the Gate group on the Gate Tools tab of the ribbon Alternatively right click the gate and select Remove from the context menu You can also delete a gate using the Delete key on the keyboard Deleting a gate will delete all data and statistics for the gate including all child gates and plots derived from the gate Editing Gates 91 se s80 NO sishjeuy g Je deyug Displaying Statistics You can change the statistics items displayed for each
15. Plate position adjustment Apply saved custom plate position 6 Well Plate A3 B3 X mm 0 0 X mm 0 0 X mm 0 0 Y mm 0 0 Y mm 0 0 Y mm 0 0 12 Well Plate Al A4 C4 X mm 0 0 X mm 0 0 X mm 0 0 mm 0 0 Y mm 0 0 Y mm 0 0 24 Well Plate For details about each item see User Preference dialog page 105 Exchanging the Sorting Chip The sorting chip must be changed regularly to ensure an unobstructed flow path 1 Click Exchange on the Cytometer tab of the ribbon A confirmation dialog appears 2 Click Yes The Chip Exchange wizard appears 3 Scan the QR code on the packaging of the replacement chip Chip Exchange 1 Chip Detection Please scan the QR code printed on the Sorting Chip package If the scan is successful the chip information is displayed 4 Click Next Chip Exchange Vv 1 Chip Detection Chip Details If the information is correct click the Next button 2 Chip Information Detected Information Part Number J82aR1N Nozzle Size 100um Instrument Settings Sheath Pressure 20PSI Target Event Rate 5 000eps You are prompted to exchange the chip 5 Open the flip up door on the front panel and remove the current sorting chip 6 Insert the replacement chip in the chip insertion slot then close the flip up door Click Next 5 Chip Exchange Vv 1 Chip Detection Please exchange the chip first then
16. The Sort Settings dialog appears 7 Configure the settings for sorting into wells on the Plate Sort Settings tab The description in this section shows a 96 well plate as an example The operation is the same for all other multi well plates Sort Settings 96 Vjell Plate 00000000000 Close G4 If using index sorting enable the Add index sort information checkbox In index sorting you can associate the cells sorted in each well with the data points for those cells on plots For details see Index Sorting page 101 2 Select the sorting sequence in Sort Layout Settings e Column To Row Sorting occurs in the sequence Al BI C1 H1 A2 gt B2 G12 gt H12 e Row To Column Sorting occurs in the sequence Al A2 gt A3 AI2 BI gt B2 H11 gt H12 3 Select the target well to set You can select the well using the following buttons or by clicking the well on the palette I Select wells in alternate columns Select wells in alternate rows Select wells in groups of four You can select multiple wells using the Shift and Ctrl keys The ID is displayed in Sort ID You can change the value in Sort ID manually 4 Select the gate whose population you want to sort into the well in Sort Gate You can change the color of the gate in Color Select a sorting mode in Sort Mode The default is Single Cell Single Cell mode is re
17. f j 10 tf the sheath tank was refilled while the instrument is F in use click Settings on the Cytometer tab of the i ribbon to display the Advanced Settings dialog and then click Ready on the Pressure Options tab To continue sorting click Sort Calibration on the 1 Reni the tnk wishes tid Cytometer tab of the ribbon to run auto calibration soueUuslUle g Ja deyy I For details see Automatic Calibration page 46 Do not touch the surfaces of the sheath tank lid that come into contact with sheath fluid Take care not to spill any fluid Do not use a pump or other object that has not been sterilized for filling the tank It is recommended that the tank be placed immediately beneath the tap of the source sheath fluid container so that the tank is filled using gravity alone Overfilling the sheath tank may damage the unit Do not fill the tank beyond the full line inside the tank Refilling the Sheath Tank 147 soueuajUleW g sajdeyy 148 Emptying Changing the Waste Tank Check the fluid level in the waste tank daily before starting If itis full dispose of the waste fluid or replace the tank with a new tank Also the waste fluid must be disposed or the waste tank must be replaced when prompted to do so in SH800 Software When handling the waste tank always follow the laboratory rules for the handling of waste fluids Waste fluid may contain virulent or hazardous substances that may
18. ssc 30 0 F 638nm Off Turn On A wl Le 2 Ls P T FLA 4 FL 40 0 Temperature Controi j inne BEES r aa eel be Sample 5 Wa C FL3 BU TS 2 FLA V0 S Z 5 x ls nc x Sle c ds An OA ELA 7 Collection 5 Y C FL5 40 0 70 i LO 40 0 i Skip rinse outside of sample probe with DI water Advanced Settings As Review the instructions in the Operator s Guide before changing options in Advanced Settings Laser Turns each laser on off Temperature Control Sets the control temperature of the sample loader and collection stage For details about each item see Temperature Control Settings dialog page 120 Threshold Sets the threshold trigger for detecting events e Channel Selects the channel FSC BSC FL1 to FL6 used to trigger the detection of events e Value Sets the input threshold signal level of the trigger channel as a percentage of the maximum signal from the detection module Sensor Gain Sets the gain of each detector e FSC Selects the gain of the forward scatter detector The gain setting has a 16 step range e BSC FLI to FL6 Sets the gain of the corresponding back scatter and fluorescence photomultiplier tube PMT detectors as a percentage of the maximum level Probe Wash Sets probe wash options To skip cleaning of the outer surface of the sample probe using DI water from the DI water tank when cleaning the probe place a check mark in the
19. Click Settings on the Cytometer tab of the ribbon The Cytometer Settings dialog appears 2 Click Advanced Settings The Advanced Settings dialog appears 3 Click Standby on the Pressure Options tab 4 Check that Standby is displayed on the LCD monitor When Standby is displayed disconnect the fluidics cart air line clear tubing from the top of the sheath tank For details see Refilling the Sheath Tank page 146 Do not force the lid down or place place objects on the lid or move the tank while the tank is pressurized The sheath tank and sheath fluid line must not be disturbed to ensure stable measurement conditions Position the sheath fluid line where it will not be affected by drafts from air conditioning or vibration of the air compressor Name and Function of Parts O Lock Locks the tray The lock prevents the tray from being withdrawn To lock the tray use a coin or similar object and turn to the LOCK position To unlock turn to the UNLOCK position Tray Holds the sheath tank DI water tank and waste tank and can be withdrawn when performing maintenance The tray is fitted with a lock to prevent it from being withdrawn accidentally See Maintenance page 143 Adjustable feet Feet are adjustable to ensure the fluidics cart does not move during data acquisition Cart connection cable connector MAIN UNIT Connects to the cart connection cable connector
20. G Inserted at an angle Attach the IN and OUT connectors to the appropriate ends of the filter 2 Attach the filter spacer and cap Do not forget to add the filter spacer 3 Lower the flip up door slowly until it can support its Ejecting the Sorting Chip own weight Manually The sorting chip is ejected automatically when you click Unload in SH800 Software However if for any reason the chip will not eject normally the chip can be removed manually 1 Open the flip up door on the front panel to access the sorting chip and hold it in position 4 With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel 5 Grasp the sample line connector firmly and pull it out towards you to the position if not in position already 5 fA Sul Pulling the sample line connector withdraws the sorting chip connector interface allowing the chip to If the sample line connector is out at the front go to be removed step 6 6 Eject the chip by pushing the top of the thumbwheel from front to back If the sample line connector is not out at the front go to step 2 soueuaule g seideyo I 2 Turn off the compressed air supply Have an assistant help if you cannot reach the air supply control valve while holding the flip up door Note open Touching the thumbwheel or the sample line will directly affect the chip alignment If you touch either and then op
21. Power supply error for peltier Circuit error on the LD3 board Error Messages GUI Error Message Recommended Solution Shut down the cytometer then try again Temperature error of the 561nm laser Error on the eeprom The ambient temperature is too low Communication error on the PCT board Load Chip Error Failed to hold the chip Please reinsert the chip Load Chip Error Failed to detect the chip Please reinsert the chip Leakage is suspected in the collection area Leakage is suspected in the drip tray Failed to start feeding sheath fluid Failed to fit the chip If the problem persists contact your Sony distributor for service The cytometer may be overheating Shut down the cytometer and allow the temperature to fall within the rated range before rebooting If the problem persists at temperatures not exceeding 35 C 95 F contact your Sony distributor for service Shut down the cytometer and wait a while before trying again If the problem persists contact your Sony distributor for service The cytometer may be too cold Shut down the cytometer and allow the temperature to rise within the rated range before rebooting If the problem persists at temperatures exceeding 10 C 50 F contact your Sony distributor for service Shut down the cytometer and wait a while before trying again If the problem persists contact your Sony distributor for service Insert the ch
22. Sensor Gain Specifies the sensor gain of the detectors FSC Specifies the gain for the forward scatter detector The gain setting has a 16 step range BSC Specifies the gain of the back scatter photomultiplier tube PMT detector as a percentage of the output signal of the detector The default value is 30 0 FL1 to FL6 Specifies the gain of the corresponding fluorescence channel photomultiplier tube PMT detector as a percentage of the output signal of the detector The default value is 40 0 e Only the gain of fluorescence channels that have pulse parameters selected for acquisition can be adjusted The gain settings of other channels are disabled e If the PMT gain is too high the detector output circuit may become saturated providing incorrect signal output In this case reduce the gain and then readjust the instrument settings to find the optimum operating point Configuring Detector Settings 19 sluewuedxy Buunbyuoy p Jaldeyy iii s yu wn dx Buunbyuog p Ja deyD 80 3 Click X to close the dialog The settings are saved The gain parameter values directly affect the fluorescence compensation calculation of the spillover matrix Do not change the gain of the channel detectors after or while recording the control tubes in a compensation panel The fluorescence compensation process is valid only for the instrument settings in place when all control tubes in a compensation panel are recorded If
23. Sorting Mode 93 uos 9g Jajdeuy 94 The SH800 supports the owns eight sorting modes for obtaining the desired sorting results The modes provide different levels of purity and yield Note that the droplets that are sorted in each sorting mode vary depending on the size of the nozzle of the sorting chip Single Cell Increasing purity Increasing yield rans Cell Ultra Purity free Semi Purity Normal Semi Yield Yield Ultra Yield page 95 page 97 page 97 page 96 page 96 page 98 page 98 page 99 sorting Number of droplets sorted 130 um sorting chip A DSD ODODO SO ODO MaRDOMCD M a a Number of droplets sorted Sorted droplet Waste droplet Multi drop inclusion Multi drop exclusion Sorting regions 1 Purity measures the proportion of target cells collected as a percentage of all cells collected 2 Yield measures the proportion of target cells collected as a percentage of target cells in the sample It indicates the percentage of the target population that is collected A high degree of purity of targeted cells can be obtained if the targeted cell events occur in isolation from other unwanted events In this case the droplets containing the targeted events are sorted while all unwanted events and empty droplets are passed to waste disposal In practice Sorting Mode ANA l ANA l GS Ge gt a 1 TED JAD DPO sun ene
24. Target cell is in lt Not sorted main droplet and X 7 Conflicting cell is in nearest edges of ia A nearest edge of adjacent droplets are adjacent droplet empty 130 um sorting mode A droplet is sorted 1f it contains one or more targeted events of a single type and the nearest edge region of both of the adjacent droplets is empty or non conflicting In addition if the targeted event is near the edge of the droplet the nearest adjacent droplet is also sorted if the nearest edge region of the subsequent droplet is empty or non conflicting One or two droplets are sorted for each event matching the sorting criteria Sorted Target cell is in main droplet and adjacent droplet and nearest edge of subsequent droplet are empty 4 Not sorted Conflicting cell in nearest edge of subsequent droplet above cell is in nearest edge of adjacent droplet Purity Mode In Purity mode cells are sorted with high purity 100 um sorting chip A droplet is sorted if it contains one or more targeted events of a single type and the nearest edge region of both of the adjacent droplets is empty or non conflicting One droplet is sorted for each event matching the sorting criteria H lt 130 um sorting chip A droplet is sorted if it contains one or more targeted events of a single type and the nearest half of both of the adjacent droplets is empty or non conflicting In addition if the targeted event
25. When the sample line has been changed the login screen reappears Changing the Sample Loader O Ring Changing the Sample Loader O Ring The sample loader is fitted with an O ring in the base of the injection chamber that seals the chamber when the sample tube is lifted into the chamber The O ring should be cleaned regularly and replaced every 3 to 6 months to ensure the integrity of the seal 1 With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel 2 Open the flip up door on the front panel to access the sample probe and hold it in position Lae gt 3 Turn off the compressed air supply Have an assistant help if you cannot reach the air supply control valve while holding the flip up door open 4 Lower the flip up door slowly until it can support its own weight 5 Remove the sample probe The sample probe is a precision component It should be removed so that it cannot be damaged accidentally while removing the O ring For details see Changing the PEEK Sample Line PEEK sample line compatible models page 169 or Changing the Sample Line and Probe Non PEEK sample line compatible models page 172 6 Push the bottom of the sample loader door in slightly and then raise the door and hold it open If a sample tube is currently in the sample loader remove it 7 Push the sample loader waste catcher white into its recess to provi
26. Zip Code Country Institution Enter the name of the institution Address Enter the street address of the institution City Enter the name of the city or town State Enter the name of the state or province Zip Code Enter the postal code number Country Enter the name of the country or region OK Applies the settings and closes the dialog Cancel Cancels the settings and closes the dialog User Preference dialog The User Preference dialog is used to specify software preferences unique to each user User preferences are loaded automatically when a user logs in saving having to configure the instrument you want to perform acquisition analysis or sorting The User Preference dialog is displayed by clicking Information gt User Preference on the File tab of the ribbon Tip You can click Reset to restore the default values for each setting Defines default settings related to sorting User Preference Sort _ Define default settings for sort control Reset Cytometer Sort Settings Experiment eclintondt oO itinerant method Compensation 2 oa Tubes User Defined Color Plate Position FCS File Plate position adjustment Apply saved custom plate position Sort Settings Specifies the default sorting configuration Sort method Selects the sorting method The available options vary depending on the model Plate Position Adjusts the position of wells for each type of plate
27. bi all ee Hew New Creates a new blank sample group in the experiment selected in the Experiment Explorer Delete Deletes the sample group selected in the Experiment Explorer Duplicate Duplicates the sample group selected in the Experiment Explorer Save as Template Displays the Save as Template dialog page 115 for saving the sample group selected in the Experiment Explorer as a template used when creating a new experiment Apply Template Displays the Apply Template dialog page 115 for selecting a template for a new sample group selected in the Experiment Explorer Main Window Tube group Contains tool buttons for creating deleting and performing other actions on sample tubes rid qr i ty ma v ix Wy lip E 3 New Delete Duplicate Save as Ar Open Apply Template Tempsie Worksheet _ Compensaton New Creates a new blank sample tube in the sample group selected in the Experiment Explorer Delete Deletes the sample tube selected in the Experiment Explorer Duplicate Duplicates the sample tube selected in the Experiment Explorer Save as Template Displays the Save as Template dialog page 115 for saving the sample tube selected in the Experiment Explorer as a template used when adding a sample tube Apply Template Displays the Apply Template dialog page 115 for selecting a template for a new sample tube selected in the Experiment Explorer Open Worksheet
28. dialog page 128 for exporting the data in the Gates and Statistics table as a CSV format file Main Window Alignment group Contains tool buttons for displaying a grid on the worksheet and for aligning displayed items with the grid O HH fre He Show Snapto Auto Grid Grid Arrange Alignment Show Grid Displays the grid on the worksheet Snap to Grid Aligns the plots and Gates and Statistics table on the worksheet with the grid when they are moved or added Auto Arrange Automatically aligns the plots and Gates and Statistics table on the worksheet to the grid Worksheet View group Contains tool buttons related to the display of items on the worksheet and for saving the current settings E QA R HO ris he Change 100 Palette to Favorite Settings I 1 i Worksneet View Change Palette Displays a drop down palette for changing the display style of the selected plot mg l sa rna E par pii m n E Q R CJ Sev EQ E o Change 100 Favorite Copy Exportte Show Analyze Apply Ma Palettes Settings Picture CSVFile Magnifier Index Data Compensation Compe 1 oat e Black background e Black background with highlighted outlying events e White background e White background with highlighted outlying events Highlighting outlying events is useful for identifying and analyzing rare events Zoom Displays a drop down menu for changing the display zoom factor of th
29. gate in the Gates and Statistics table For details about statistics see Gates and Statistics Table page 131 1 Click Edit Statistics in the Statistics group on the Worksheet Tools tab of the ribbon Alternatively right click the Gates and Statistics table and select Open Statistics Editor from the context menu a E orksheet Tools Plot Tools Gate Tools 1 Data Source 1 All Events B ca C Es te amp Q ay B ii Remove Edit Show Export Statistics Show Snap to Gate Gate Table Statistics to CSV File Grid Grid The Statistics Editor dialog appears Specify the items to be displayed in the Gates and Statistics table Selected Gate A FSC A FSC H BSC A BSC H Brilliant Violet 421 A Brilliant Violet 421 H FITC A FITC H PE A PE H APC A APC H PerCP Cy5 5 A PerCP Cy5 5 H PE Cy7 A G Select a gate The statistics values for the population in the selected gate are displayed in the lower area 2 Place a check mark in the checkboxes for the Statistics you want to display for each parameter 83 Click Apply to update the statistics displayed in the Gate and Statistics table 4 Click Close to close the dialog Displaying Statistics To change the width of columns Drag the right edge of a table column to change the width of the column To change the display order of columns Select the title of the column to move and dr
30. modified before during or after data acquisition Marker names and fluorochrome names are optional but recommended If names are not specified the fluorescence channel numbers FL1 to FL6 will be displayed as the axis labels on plots by default providing limited feedback as to the type of data displayed on plots 3 Click Apply or OK to apply the measurement settings Clicking OK applies the settings and closes the dialog Editing an Experiment Editing an Experiment You can modify an existing experiment as required You can also edit an experiment using the Experiment tab of the ribbon For details about tools available on the Experiment tab see Experiment Tab ribbon page 113 Editing a Sample Group You can add delete and perform other edit functions on a sample group in an experiment Edit operations are performed using buttons in the Sample Group group on the Experiment tab of the ribbon You can also perform the same operations by right clicking a sample group in the Experiment Explorer Worksheet Took Tube 2 Data Source 1 Compensation For details about each button see Sample Group group page 114 onthe Experiment tab of the ribbon Editing a Sample Tube You can add delete and perform other edit functions on a sample tube in an experiment Edit operations are performed using buttons in Tube on the Experiment tab of the ribbon You can
31. on the Cytometer tab of the ribbon as required to fill the DI water line with DI water Changing the PEEK Sample Line PEEK sample line compatible models The PEEK sample line must be replaced regularly to prevent the buildup of salts restricting the sample flow The PEEK sample line should be checked for any signs of damage or wear A damaged PEEK sample line may split and cause sample fluid leakage The PEEK sample line does not support autoclaving however the probe adapter does support autoclaving Always leave the PEEK sample line connected when the unit is not in use for protection against dust Use the following procedure to attach the PEEK sample line There is a risk of fluid leakage if the sample line is not attached correctly For details see Changing the Probe Adapter PEEK sample line compatible models page 175 Tip Sample fluid may contain biological chemical or other agents Always wear biology laboratory gloves and other protective clothing mask and goggles when changing the PEEK sample line 1 In SH800 Software click Sample line on the Cytometer tab of the ribbon 2 Click Start to begin changing the PEEK sample line e Always follow the instructions displayed in the wizard when changing the PEEK sample line e The sorting chip must be replaced or reinserted after changing the PEEK sample line The PEEK sample line can also be changed using Sample line exchange on th
32. optical filter pattern 2 ELZ 62560 FL2 62560 FL 60060 FLA 6650 FLT 62560 FLS 61780 FLA 66580 FLA 66550 PE Cy5 5 FL5 720 60 Fluorochrome Detection Matrix 2 lasers 488 and 561 nm lasers optical filter pattern 1 Fluorochrome Channel Pee ever etre ser P Propidium lodide FL3 617 30 7 AAD 7 Aminoactinomycin D FL4 665 30 PE Cy5 670 FL4 665 30 PerCP Peridinin chlorophyll protein 675 FL4 665 30 shee Moroney a8 cc G FL5 720 60 Fluorochrome Detection Matrix sno ueLjj v s y g X pu ddy 207 sno ueLjj s g xlpueddy 208 3 lasers 405 488 and 638 nm lasers optical filter pattern 2 600 60 ee wr xw xr Nr il ams os ON x ee es Nes es me 488 nm laser FL2 525 5 665 30 _ N AN AN A il a xr ee ee ur ee ee Su PE Cy5 5 FL5 720 60 PerCP Cy5 5 FL5 720 60 x xr r w ee Sur O APC Alexa Fluor 750 FL6 785 60 Ae Fluorochrome Detection Matrix 3 lasers 405 488 and 561 nm lasers optical filter pattern 2 Fluorochrome Channel tauontweor e e A OO o T 8 C A OO a A FL 25 50 C e A NO C FL 62560 Brillant Vio 660 fe FLA 66570 a a Ie Seen o o i e a peemewn r ooo ew e 09 a e 000 FARO FArioacinonya BY eer Pegs ooo y PorGP Perinin chorph pren fes rees poss o e ROC Peeoss o y e
33. optical filters BPF Dichroic longpass optical filters reflect light below a certain wavelength and transmit light above a certain wavelength Bandpass optical filters transmit light in a narrow band of wavelengths and block light of all other wavelengths For bandpass optical filters the first number in the name indicates the center wavelength and the second indicates the bandpass width e g 525 50 is a 525 nm bandpass optical filter with 50 nm bandwidth Optical System Longpass optical filter LPF beam splitter c xe U 2 E Cc H aS Reflected Passband 50 Optical filter Wavelength wavelength Bandpass optical filter BPF c 2 N Q Center wavelength aS Stopband Passband Stopband 50 Bandwidth Wavelength Forward scatter light passes through a collimator and a bandpass optical filter BPF and is detected by a photodiode PD The bandpass optical filter determines the wavelength of the laser source for which forward scatter is measured The SH800 is configured by default to measure forward scatter light at a wavelength of 488 nm Back scatter light passes through a bandpass optical filter and is detected and amplified by a photomultiplier tube PMT detector The bandpass optical filter determines the wavelength of the laser source for which back scatter is measured The SH800 is configured by default to measure back scatter light at a wavelength of 488 nm
34. other droplets containing non target cells are uncharged and collected in the waste catcher The target cells for sorting are specified using gates in SH800 Software The trajectories of droplets that are sorted in the collection tubes are called side streams For details see Side stream monitor page 136 The deflection plates are charged at an extremely high voltage Contact with charged deflection plates can result in serious injury or electrical shock Do not touch the deflection plates while power is connected to the main unit Side stream calibration and data acquisition can be adversely affected by bright light shining directly on the side stream monitor Do not place the front of the instrument near sources of bright light such as lamps or unshaded windows Waste catcher Collects the uncharged non target droplets The collected droplets are passed to the waste tank in the fluidics cart Collection tubes Collects the electrostatically charged target droplets The SH800 supports the following collection tube devices Each type of collection tube is used with a corresponding holder that mounts on the collection stage Collection tubes 15 ml conical tubes Collection tube holder 15 ml 5 ml round tubes Collection tube holder 5 ml 8 well strips 8 well strips holder Slide glass Slide glass holder 6 well plates 96 well plate holder 12 well plates 24 well plates 48 well plates 96 well pla
35. roneo wa WN M Q Je deyD 24 Waste fluid may contain biological chemical or other agents Always wear gloves and other protective clothing mask and goggles as required when emptying or replacing the waste tank The handling of waste fluid should be performed in accordance with biological hazard handling safety procedures Ethanol tank Supplies the cleaning fluid ethanol used to clean the fluidics system during cleaning cycles A sensor detects the ethanol level for display on the LCD monitor and host computer An alarm is generated when the ethanol falls below a set level The tank has a capacity of 5 liters 1 3 gallons US Sheath tank Supplies the sheath fluid used as the flow medium for transporting the sample fluid in a laminar flow through the sorting chip and into the collection tubes The tank is pressurized using regulated compressed air supplied from the main unit A cart sensor detects the sheath fluid level for display on the LCD monitor and host computer An alarm is generated when the sheath fluid falls below a set level The tank has a capacity of 10 liters 2 6 gallons US Never use the supplied sheath tank for anything other than the intended purpose When opening the sheath tank cap check that Standby is displayed on the LCD monitor or that the power to the main unit is turned off If Standby 1s not displayed place the unit in Standby mode using the following procedure G
36. systems include e Sorting chip loading and alignment e Sample tube loading e Collection tube loading and alignment e Fluorescence compensation e Droplet formation frequency and calibration e Sample line and sorting chip cleaning e Temperature control of sample tube and collection tubes 1 In this document sample tubes 8 well strips slide glass and multi well plates used for collecting sorted samples are collectively referred to as collection tubes Manual controls for fluorescence compensation and droplet formation are also supported in SH800 Software The hardware user interface is designed to be as simple as possible to use An LCD monitor on the front of the unit displays status information and notification of error messages The SH800 connects to standard 100 V to 240 V AC 50 60 Hz power supplies and has a 17 5 C to 27 5 C 63 5 F to 81 5 F recommended operating temperature range It communicates with the host computer via the PC connection cable The SH800 requires the connection of a laboratory compressed air supply for operation It can also be supplied using an external air compressor unit An external fluidics cart placed under or near the main unit supplies the main unit with sheath fluid used during testing and ethanol used for cleaning and collects the waste fluid from the main unit A single host computer running SH800 Software controls all aspects of instrument operation including cel
37. tab of the ribbon You can perform the same operation from the context menu by right clicking the plot Poi y sil gt File Experiment Cytometer Fa Densit gt pes lds a By J bB z Dot Piot New New Dot New Remove Duplicate T Density Piot Histogram Plot Piot lki Histogram The selected plot is duplicated Compensation 2 Edit the duplicated plot as required using the buttons on the Plot Tools tab of the ribbon You can perform the same operation from the context menu by right clicking the plot For details about each button see Plot Tools Tab ribbon page 129 Removing a Plot Select a plot to be deleted on the worksheet then click Remove Plot in the Plot group on the Plot Tools tab of the ribbon Changing Axis Scales Select a plot on the worksheet then click the desired scale for each axis in the Scale Type group on the Plot Tools tab of the ribbon M Worksheet Tools Plot Toois neter Compensation Tube 2 Index 96 Well Data Source 1 All Events D Density es Dot Plot 42 Duplicate Auto Adjust Auto Adjust Piot dda Histogram pe X X Axis You can also change the scale by right clicking the plot and selecting from the context menu All Events 1000 K 800 A Linear Log BSC A Biexponential I Auto Adjust Axis Default Adjust Properties F4 0 200 400 600 800 FSC A 1 000 Editing Plot
38. using existing templates and or recent experiments 1 Ifthe Create Experiment window is not displayed click New on the File tab of the ribbon 2 Create an experiment Select a template Clicking a template or an experiment on the left displays the structure of the selected experiment on the right side of the window For details about changing the structure of an experiment see Creating a Customized Experiment page 70 Enter a name for the experiment in Name You can configure sample groups sample tubes pulse parameters and other settings for data acquisition For details about each item see Create Experiment Window page 111 3 Click Create New Experiment If Blank Template was selected the New Experiment Startup Procedure dialog appears Proceed to step 3 If an existing template or experiment was selected the main window appears See Acquiring Data page 49 Creating an Experiment 47 uoneiedo oiseg Jaldeyuy Kii uoleliado olseg E Jaydeyy 3 Select the action to perform then click OK New Experiment Startup Procedure This new experiment is generated from blank template please select the first process and click OK button 44 Start acquiring first tube v 2 in case that you don t have control samples for compensation Start compensation wizard CU in case that you have contro samples for compensation yu can set the def
39. when sorting cells larger than 15 um in Normal mode The side streams may become unstable when sorting cells of size 20 um or larger If droplet marks are visible on the collection tube holder after sorting wipe clean with a lint free soft clean cloth sprayed with ethanol and then another moistened with water Side stream monitor Displays the image from the side stream monitor camera Left side stream Right side stream For details about adjusting the deflection angle of side streams see Advanced Settings dialog page 122 To Sort Selects the gates for the target events to sort into the left and right collection tubes Stop Value Specifies the event count conditions to stop recording automatically for each of the left and right collection tubes You can select a value or enter a value from the keyboard Entering a value of 0 disables the stop condition Sort Statistics pane Displays the statistics about events during sorting Elapsed Time Displays the cumulative elapsed time since the start of sorting Remaining Time Displays the estimated remaining sorting time Sort Count Displays the number of events sorted Sort Rate Displays the rate at which events are detected during sorting in units of events per second eps Sort Efficiency Displays the number of sorts attempted as a percentage of targeted cells in the sample Abort Count Displays the number of events that
40. window Clicking displays a calendar for specifying a date Event Rate Experiments From Di Keyword t Q 2 Click amp to set optional addition search options a JE 5 Keyword a Sample Ga Search Settings amp Sample Sort by Experiment Measur 1 Oldest to Newest Compe o 4 ff Compe Negi Brilli g mc OPE i APC Sample Group Information b PerG OPEC Newest to Oldest AtoZ PN N gt Ob 6 ice oA Search Condition wv Experiment Information Tube Information ube 53 Tub Oldest to Newest Displays the search results in chronological order from oldest to newest Newest to Oldest Displays the search results in chronological order from newest to oldest A to Z Displays the search results in ascending alphabetical order Z to A Displays the search results in descending alphabetical order Search condition Selects the experiment components to search for the entered text keyword Reset Resets the search options to their defaults Experiments matching the search criteria are displayed in the Experiment Explorer Saving Tube Dataas FCS Files You can export data recorded for tubes in flow cytometry industry standard FCS 3 0 or FCS 3 1 format for use in third party applications SH800 Software does not support the importing of FCS format files 1 2 Select the target tube in the Experiment Explorer on the Experiments ta
41. 1 e Screws 2 AMS evacuator installation should be performed by service personnel However you can attach and remove of the vacuum tubing by yourself as follows Connecting the vacuum tubing 1 Verify that the unit and the evacuator are turned off 2 Attach one end of the vacuum tubing to the HEPA filter bracket Place a hose clamp on the vacuum tubing and attach the tubing to the HEPA filter bracket HEPA filter bracket Vacuum tubing 2 Secure the hose clamp using a Phillips screwdriver Hose clamp b 3 Attach the other end of the vacuum tubing to the AMS evacuator Place a hose clamp on the vacuum tubing and attach the tubing to the AMS evacuator s tube joint 2 Secure the hose clamp using a Phillips screwdriver Connecting an AMS Evacuator SH800Z and SH800ZP snosueljaosi g xlpueddy 213 sno ugjj siy g xipueddy 214 Vacuum tubing Hose clamp Tube joint 4 Turn on the evacuator at the evacuation level specified during installation 5 Turn on the unit e The evacuator must be turned on when performing automatic calibration on the unit If the evacuator was not turned on turn on the evacuator before performing automatic calibration again e Position the vacuum tubing so that it is not in contact with the unit fluidics cart and sheath line Disconnecting the vacuum tubing Perform the procedure for connection in reverse Connecting an AMS Evac
42. 100 00 0 00 PE Cy7 0 00 0 00 0 00 000 0 00 100 00 s Use Negative Value for Compensation if you use different type sample e g comp beads from real target sample please uncheck the checkbox The spillover matrix and the negative fluorescence compensation values are calculated and then displayed 1 8 Check the compensation matrix values Compensation Wizard check the spillover matrix value If they are ok please click Finish button vV 1 Gain Settings z Vv 2 Acquiring Sample Data S gt j i a Brilliant Violet 421 100 00 lie 2 E G 2 APC S PerCP Cy5 5 PE Cy7 19 Click Finish to exit the Compensation Wizard 64 Flushing the Fluidics System Flushing the Fluidics System When measuring samples the sorting chip sample line and sample probe should always be flushed with sheath fluid after unloading a sample tube and before loading the next sample tube to eliminate carryover contamination between samples Check that data acquisition and recording are stopped The fluidics system cannot be washed during data acquisition 2 Click Probe Wash in the Cleaning group on the Cytometer tab of the ribbon iment Cytometer Compensation Tube ae a is H 2 G w amp Fa e A ia alt HCIO Di Collection Sampie Bleach DI Shutdown Oeaning Rinse Rinse Legit eat ae The sorting chip sample line and sample probe are flushed with sheath fluid For details
43. 2 Next Tube Status Ready Elapsed Time 00 00 00 0 Total Event 0 Event Rate 0 eps apsed Time 00 00 00 Q vent Count 0 100 000 Co v p Condition Event Count 4 E Sample Group 1 Sample Group Information gt Measurement Settings Compensation Settings Compensation Panel 4 Tube 1 1 Tube Information gt Worksheet Settings gt Stop amp Sort Settings ay 2 Way Data Source i 4G Tube 2 1 Tube Information Worksheet Settings Stop amp Sort Settings Acquisition Experiments Currently assigned sample tube and sample group Next Tube Assigns the next tube If there are no further tubes it duplicates the current tube and assigns the new tube Instrument status Displays the status of the instrument The following states are displayed e Initializing e Standby e Ready e Pause e Measurement e Probe Wash e No Chip e Chip De bubble e Ethanol Cleaning e Error e Waiting e Bleach Cleaning e DI Rinse e Shutdown Main Window Data acquisition status Displays the status of data acquisition Elapsed Time Displays the cumulative elapsed time since the start of data acquisition Total Events Displays the total number of events detected during data acquisition Event Rate Displays the rate events are detected during data acquisition in events per second eps Range 0 eps to 20 000 eps The event rate indicator on the
44. 5 vitc 00 m 0 3 Unit Celstus v Advanced Settings Ay Review the instructions in the Operator s Guide before changing options in Advanced Settings The Advanced Settings dialog appears Advanced Settings wo Options Stream Options Opt ev p Drive r p Stage D lock Z Ax X 1 Sort Delay a Z Step 1 l Sort Phase 0 g a gt Back to Original point Ss Correct v e A Camere h Calibration Display Mode A 048 Save Droplet Info LED Yv Control Breakoff sf Sample Empty Detection Step 1000 age ollec Back A d lt gt n Deflect ting Stream Left Waste Stream rt gi v For details about each item see Advanced Settings dialog page 122 Adjusting Sort Parameters Manually Experienced Users 103 uoNduoseg mopuiM Z Je deuyD 104 Window Description Chapter e The menus and buttons displayed vary depending on the user account privileges e The screenshots displayed in this document may vary from the actual software File Window The File window Backstage View is displayed by clicking the File tab of the ribbon For details about Restart Logout and Exit see File window page 26 Information window The Information window is displayed by clicking Information in the menu on the left side The Information window is used to change login passwords set institution information and add modity user accounts Example Administrator
45. 96 Well Plate v Sas Sort Settings Sorting 59 uoneiedo oiseg g13 deyo HIN uoleisdo olseg sa deyuyD e o 0 60 Tip Starting sorting automatically starts sorting and recording Basic sorting statistics are displayed together with progress bars if an automatic stop condition 1s Specie Sort Statistics Total Elapsed Time 00 00 00 Sort ID eS OSooeoeoes Elapsed Time 00 00 00 Total Progress 0 2 Well Number qeoeeeooeoeeee Remaining Time 00 00 00 meeeeceosceoe F Sort Mode Sort Count 0 Cell Size j Sort Rate 0 eps ree feos sosoocooo 5 Sort Gate ASS Te SWT Sort Efficiency 0 Stop Count Abort Count O Oeps Double clicking the image displays the side streams in a Separate window for monitoring sorting Droplet Viewer Q x075 Double clicking the plate image or right clicking and selecting Sorting Monitor from the context menu displays the Plate Sorting Monitor window for monitoring progress You can also display the window by right clicking a tube in the tube list and selecting Show Sorting Report from the context menu Plate Sorting Monitor Sorting Method 96 Well Plate Sort Statistics Total Elapsed Time 00 11 11 Total Progress 96 96 TE EER lt lt EE 100 se i ae SE ae m D E a a a on C E ar a He fe ae a a a
46. A i 0O c 0O D Well Select Mode Select this mode when you want to display the position on plots of cells sorted in each well You can select multiple wells at the same time Gate Select Mode Select this mode to display only those wells encompassed by the selected gate You can also select wells to display the position of cells in those wells on plots If Well Select Mode is selected Displays all wells in the multi well plate Clicking a well in the dialog displays the position on plots of the cells collected in that well Placing a check mark in Show All Events displays all data acquired during sorting includes events that were sorted and events that were not sorted on plots Index Sorting Specifies the size of the dot displayed on plots indicating the data position Index Analysis Analysis Mode Well Select Mode v v Show All Events Show Cross Lines g2 A B C D E F G H 1 2 3 4 5 6 7 8 9 0 HW 2 All Events To view the dot position accurately Placing a check mark in Show Cross Lines displays horizontal and vertical lines intersecting the dot position for more accurate positioning Tip Clicking amp displays the User Preference dialog page 105 for setting user default preferences If Gate Select Mode is selected Displays only those wells that contain events encompassed by the gate selected in Selected Gate Placing a check mark in Overlay Index Events displays t
47. Apply saved custom plate position Applies saved plate position adjustment values Cytometer tab Defines default settings related to the instrument User Preference Pe 2 __ Define default settings of cytometer control Reset Cytometer HEaren Lrcars Leone aver eran ervey Agitator Experiment Set default agitator properties after user login Worksheet A A MG Agitator Compensation On Off User Defined Color Temperature Control FCS File i Set default sample temperature setting after user login Sample Temperature Control Off On 5 C 41 F On 37 C 98 6 F i Set default collection temperature setting after user login wy Collection Temperature Control Off On 5 C 41 F Probe Wash Set default probe wash setting after user login Skip rinse outside of sample probe with DI water OK Cance Agitator Agitator Selects the default status of the agitation unit Temperature Control Sample Temperature Control Selects the temperature of sample fluid in the sample loader e Off e On 5 C 41 F e On 37 C 98 6 F File Window uondos q mopuiM Z JeIdeyD I 105 uolduoseg mopuiM Z Je deuyD 106 Collection Temperature Control Selects the temperature of sorted fluid on the collection stage e Off e On 5 C 41 F Probe Wash Skip rinse outside of sample probe with DI water Sets whether to skip cleaning of the outer surface of the sample probe using DI wa
48. Change Palette Displays a drop down palette for changing the display style of the selected plot For details see Change Palette page 126 x1 0 x1 5 x2 0 Displays the plot at 100 default 150 and 200 size respectively Main Window uondos q mopuiM Z JeldeyD I 129 uolduoseg mopuiM Z Je deuyD 130 Property Window dialog The Property Window dialog is displayed by clicking the dialog launcher in the Scale Type group or Axes group on the Plot Tools tab of the ribbon You can also display the dialog by right clicking the worksheet plot gate or a Gates and Statistics table cell and selecting Properties from the context menu The Property Window dialog is used to change the attributes of plots and to specify the scale type and range of axes Property Window Plot Properties Linear Gate Name Displays the name of the gate required You can edit the name as required Note that the names of gates must be unique Gate Color Selects the color of the gate on plots Show Population Selects the population to display on plots Axes Specifies the scale type and range of the X and Y axes e Scale Selects the scale type e Maximum Value Specifies the maximum value of the scale e Minimum Value Specifies the minimum value of the scale Close Closes the dialog Main Window Gate Tools Tab ribbon The Gate Tools tab of the ribbon has the fol
49. Delete All Data I want to delete all data File Window uondos q mopuiM Z JeldeyyD I 109 uoNduoseg mopuiM Z Je deuyD 110 I want to delete all data Place a check mark in the checkbox to confirm you want to reset the database Delete All Data Resets the database deleting all data Close Closes the dialog Help Window The Help window is displayed by clicking Help in the menu on the left side The Help window is used to display the Operator s Guide this document and to check software version information f Open Manual EEE st1800 Software Open the Operator s Guide Version AD ME Open Manual Displays the Operator s Guide SH800 Software Displays the SH800 Software version information Clicking End User License Agreement displays the license agreement Cytometer Version Displays version information for instrument components Log Window The Log window is displayed by clicking Log in the menu on the left side The Log window is used to display the chip alignment log sort calibration log and user usage log BG Chip Alignment Log PA Sort Calibration Log Po User Usage Chip Alignment Log Displays the position alignment log for the sorting chip Sort Calibration Log Displays the sort calibration log User Usage Displays the user usage log File Window Chip alignment log Displays the time and date when the sorting chip was
50. Fluid Flow s jd uldq Buryessdo y xipueddy 195 sgjdiouudg BuryesodoQ y xipueddy 196 The formation of droplets at the breakoff point and the angular deflection of side streams are calibrated at startup using automatic setup beads For details about calibration see Automatic Calibration page 46 Optical System Optical System This section describes the signals that can be generated using the SH800 Light and Signal Generation Light Scatter When laser light hits a cell passing the optical detection point in the sorting chip light is scattered in all directions Light scattered in the forward direction is called forward scatter FSC Light scattered to the sides and back toward the light source is called back scatter BSC Sheath and sample flow Back scatter BSC Cell _ Incident laser light In general terms forward scatter can provide information about the size of the cell or can indicate the state of living cells Back scatter can provide information about the internal detail of the cell indicating complexity granularity and irregularities in a cell Fluorescence When laser light hits a cell passing the optical detection point in the sorting chip the light excitation can cause fluorochromes in molecules or antibodies that have been labeled with fluorescent markers to fluoresce in the sample producing fluorescent light FL Fluorochromes for markers emit high level fl
51. ROO Peores po o fro 7 2S 8S so So y oS FL4 PE Cy7 FL6 785 60 710 DsRed Monomer tdTomato FL3 600 60 610 r C IG FL3 600 60 Fluorochrome Detection Matrix sno ueLjj s g X ipu ddy 209 sno ueLjj s g xlpueddy 210 3 lasers 405 561 and 638 nm lasers optical filter pattern 1 Channel FLI 825 50 FL1 825 50 PE R Phycoerythrin 576 PeteasRed SS FLS 617 30 PI Propidium lodide FL3 617 30 7 AAD 7 Aminoactinomycin D PerCP Peridinin chlorophyll protein 675 FL4 655 30 FL5 72060 FL2 585 30 FL2 585 30 FL5 720 60 FL6 785 60 Fluorochrome Detection Matrix 4 lasers 405 488 561 and 638 nm lasers optical filter pattern 2 Excitation laser 405 nm laser 488 nm laser 561 nm laser 638 nm laser Fluorochrome Channel a a ce ee cameron ar an es ans E 525 50 adot 525 sa em P a te ee a a xo No No N 785 60 ews SSCS a w x x ee r lt e i a ee ee xo Noe Ne N 525 50 xo No No N Fe SR C FL3 600 60 615 FARO Arincacnonyoh O ea L PeCys dF EA PerCP Peridinin chlorophyll protein 675 FLA 685 80 PEcy5s n Pecs o o o y Fao O Ce SSSCSCS S dSSSCSCSCSCSC FO mOrange RLS 60060 DsRed Monomer 58GB 600 60 set eis Pim APC Allophycocyanin 660 Cys i O 668 ee N AN ee ee AN AN A x Ne Noe No N S Ae as oN oN
52. Sample Loads unloads the sample tube These operations are enabled only when data acquisition is paused Detector amp Threshold Settings Displays the Detector amp Threshold Settings dialog For details about the Detector amp Threshold Settings dialog see Configuring Detector Settings page 79 Sample Pressure Sets the sample fluid pressure range of to 10 Recording controls Elapsed Time Displays the cumulative elapsed time since the start of data recording Event Count Displays the number of recorded events Stop Condition Selects the stop condition for stopping data recording automatically e None e Elapsed Time Specifies elapsed time condition to stop recording automatically e Event Count Specifies the event count condition to stop recording automatically Experiment Explorer Active Experiment Displays the structure of the active experiment enabled for data acquisition recording and cell sorting For details about the Experiment Explorer see Experiment Explorer page 133 on the Experiments tab control pane Experiments Tab control pane The Experiments tab displays all current public shared and private experiments A public 0 A useri 44 EE Experiment 12 28 2013 10 33 05 PM Experiment Information a DE Sample Group 1 amp Sample Group Information Measurement Settings Compensation Settings 4 Compensation Panel a f Tube 1 1
53. Skip rinse outside of sample probe with DI water checkbox If a check mark is placed in this checkbox you should load a sample tube containing DI water after cleaning the sample probe to clean the outside of the probe Advanced Settings Displays the Advanced Settings dialog page 122 for manually adjusting sort parameters e Chip Information tab Displays information about the loaded sorting chip This is useful for checking the length of time the sorting chip has been in use Cytometer Settings Chip Type Sorting Chip Nozzle Size 100 um Part J82aRiN Chip ID 00000000 0008 0005 Year Month Date 2012 01 10 Usage 405nm 2 hours 25 minutes 488 nm 9 hours 46 minutes 561nm 0 hours 0 minutes 638nm 0 hours 0 minutes Main Window uondos q mopuiM Z JeIdeyD I 121 uolduoseg mopuiM Z Je deuyD 122 e Cytometer Information tab Displays SH800 instrument information Cytometer Settings Control Chip Information Cytometer Information Maintenance Model Type LE SH800ZFCP Serial Number 1234567890 Software Version AD ME NI 1 2 2 NI 1 2 2 CO 0x110d WF Ox110f TS 1 3 0 IM 0 2 0 JKa 0x1111 HY1 1 2 2 JKb 0x1111 HY2 1 2 2 IP 0 0 0 0 HY3 1 2 2 HY4 1 2 2 HY 5 1 2 2 IP 0 0 0 0 e Maintenance tab Contains a function to remove air bubbles from the DI water filter Click DI Filter de bubble to display the DI Filter de bubble dialog and then click Start Cytome
54. UU to Peru Abd ts 40 0 oye TA 40 0 i i a feet Hga leet Haa Hga Hia Hja 62 Performing Fluorescence Compensation 7 Adjust the size and position of the gate automatically added to the plot to encompass the desired population then click Next in the Compensation Wizard All Events 8 Adjust the gain levels of the FL1 to FL6 fluorescence channels to place the negative populations on scale in all channels You adjust the gain while viewing the histogram plots for each fluorescence channel Detector amp Threshold Settings Threshold Channel FSi ee a ac T Value 5 00 L Sensor Gain i T FSC 4 io n T BSC 30 0 3 L 40 055 40 0 40 0 40 0 PerCP Cy5 5 40 0 PE Cy7 40 0 yi The negative peaks represent the fluorescence signals detected due to cellular autofluorescence The negative peaks should be on scale and not wedged up against the axis in all channels 1 Q After adjusting the detector gain settings click Next Compensation Wizard lt lt Adjust PMT Gain settings to ensure the peak of the negative population is within the target region for each of 1 Gain Settings the gated fluorescence histograms Click Next button when complete 1 0 Click Record in the data acquisition control pane Record Restart Pause Stop The SH800 starts recording the data acquired for the negative control tube Recordi
55. View the connector from the side and check the stepped portion is not visible Check that stepped portion is not visible a a7 gt P D U T Correct Incorrect 11 Screw in the probe adapter removing any twists in the sample line then click Next 6 20 gt NY Gp t f WwW t Probe adapter ko 12 Insert the sample line into the pinch valve then click Next Make sure the sample line is inserted all the way to the back of the pinch valve e If the sample line is not correctly inserted in the pinch valve sample fluid may leak from the probe e Make sure the sample line connector is securely fastened and that the sample line is correctly inserted in the pinch valve If the sample line is not inserted correctly sheath fluid may flow in the reverse direction potentially causing serious damage to internal components and increasing the risk of biohazard contamination Be sure to insert the sample line into the pinch valve Failure to do so may result in problems caused by fluid leakage 13 Click OK to exit the Sample Line Exchange wizard When changing the sample line in maintenance mode the chip loading screen appears before exiting the Sample Line Exchange wizard Reinsert the unloaded sorting chip then click Next When the sample line has been changed the chip exchange screen appears soueUuslUlIe g Jaydeyy I Changing the PEEK Sample Line PEEK sample line compatible model
56. Way Tubes in Sorting Method A Sort Start E Unload Collection Always select the sorting method matching the cing niii Les Seo collection tube device loaded on the collection stage oe i Mode Normal wij I Regular Cell v Stop Val r vo Sort Control Basic sorting status is displayed together with progress bars if an automatic stop condition is J PAN Load Collection specified Sorting Method 2 Way Tubes Mode Normal v Regular Cell T ishini L R Elapsed Time 00 00 00 00 00 00 Remaining Time The sorting parameters vary depending on the eee 0 0 selected sorting method TEET gepi wepi Sort Efficiency 0 0 Abort Count Oi Oeps O Oeps Sorting You can monitor the side streams and droplet flow in the main window Sort Statistics Elapsed Time Sort Count Sort Rate Sort Efficiency Abort Count Side streams Remaining Time Droplet stream Oeps o O Oeps ka Dr iplet 00 00 00 i 2 Double clicking the image displays the side streams in a separate window for monitoring sorting Droplet Viewer X Q x075 nee If an automatic stop condition is configured a stop message appears when the stop condition is satisfied and sorting stops 9 Click OK to close the confirmation message dialog Tip If an event is detected that falls within both gates selected for the left and right side streams
57. a 201 Optical Filter Patterns ccssseccsssecensscennsecensseecnnees 202 Optical Filter Pattern bh onneen en cdc leiinaanerae 202 Opuical E titer PET 2 cezepctuputs asic t onstach E 203 Fluorescent Protein Optical Filter Pattern Option 203 Filter Pattern for BV PEIPL csciesicetiacrsncaesideteuastercdeaitateetedax 203 Fluorochrome Detection Matrix csccsscsessseseeeseeeees 204 Relationship between Sample Pressure and Sample Flow Event Rate ssccseseeseeseeceneesennees 212 Connecting an AMS Evacuator SH800Z and SH800ZP ce eeeseeeeeeseeeeenseeecenneeeees 213 Troubleshooting srira aoia 215 Error Messages siasii aeeai Eaa 218 Optional Accessories cccccsseeceeseeceeseesenseeeenseseeneeseees 225 Software License nnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnmnnn 225 Environmental Notices c sccsscsesssseseeseceessesseeeeeeneees 226 MOEK erase estate a ai Eea act aaa 227 6 Table of Contents The supplied installation disc includes the Operator s Guide for this unit in PDF format For more details see Using the PDF Manual page 9 Reproduction or duplication in whole or part of the software or operator s guide supplied with the unit as well as renting or leasing of the software without the authorization of the right holder is prohibited under copyright law Sony assumes no responsibility for damages loss of income
58. about cleaning manually using bleach see Cleaning the Sample Fluidics System using Bleach page 152 Shutdown Shut down the SH800 when finished measurement for the day using the following procedure The fluidics system should be cleaned according to the instructions in the Shutdown Wizard before shutting down the SH800 main unit The Shutdown Wizard describes the bleach cleaning procedure For details see Cleaning the Sample Fluidics System using Bleach page 152 Always leave a sorting chip in the loader when shutting down The chip seals the inlet ports when the SH800 is not in use 1 Click Unload Collection to unload the collection stage and remove the collection tubes Y Y Sort Control v ray Unload Collection Sorting Method Ss Sort Settings Tip Remove the splash guard when unloading multi well plates after sorting is finished 2 Clean the sample loader sorting area and the collection stage using ethanol to prevent salt precipitation within the unit and to prevent biological contamination For details about cleaning see General Cleaning page 153 After cleaning the fluidics lines are filled with DI water to prevent the formation of salt deposits Running data acquisition in this state may damage cells 3 Click Software and Hardware in the Shutdown group on the Cytometer tab of the ribbon te QO 0 Filter Hardware Software and Ethan
59. account login i Change Password 2 Institution Information 4 ecb siete File Window Example User account login ig Change Password re Institution Information Ge User Preference Change Password Allows you to change the password used to log in to SH800 Software For details see Changing Passwords page 39 Institution Information Allows you to view information on the institution using the unit You can also configure settings if you are logged in using an Administrator account Settings cannot be configured if you are logged in using a general user account Click this button to display the Institution Information dialog page 105 Account Settings Displays the Account Settings dialog page 38 for managing user accounts for SH800 Software This button is displayed for Administrator accounts only User Preference Displays the User Preference dialog page 105 for specifying software preferences which can be specified independently by each user This button is displayed for Administrator accounts only Institution Information dialog The Institute Information dialog is displayed by clicking Information gt Institute Information on the File tab of the ribbon The Institution Information dialog is used to specify information about the institution using the unit Only administrators can modify this information Institution Information Institution Address State
60. aligned together with other sorting chip information Clicking Export exports the contents of the log to a CSV format file Chip Alignment Log Sorting calibration Log Displays the time and date when sorting was calibrated together with other calibration information Clicking Export exports the contents of the log to a CSV format file Sort Calibration Log User usage log Displays the date and time users logged in to SH800 and other user information Clicking Export exports the contents of the log to a CSV format file User Usage Full Name Organization Login Date Logout Date Usage time Usage Sorting Chips Usage Cleaning Chips userl userl userL userl user Export Log dialog The Export Log dialog 1s displayed by clicking Export in the chip alignment log sort calibration log or user usage log dialogs The Export Log dialog is used to export the contents of the displayed log as a CSV format file Export Log From To Output Folder From To Specifies the interval of the log to be exported Click to enter a date Output Folder Specifies the output destination folder Click to browse for a destination folder Progress Displays the export progress Export Starts exporting the log Close Closes the dialog Create Experiment Window The Create Experiment window is displayed by clicking New on the File tab of the ribbon or New in the Exp
61. also perform the same operations by right clicking a sample tube in the Experiment Explorer SH800 Software Pict Tools ata Source 2 B wk WY DB A Rl New Delete Duplicate Save as Open Apply Copy Tempiate Te ite Worksheet Compensation For details about each button see Tube group page 114 on the Experiment tab of the ribbon Changing Component Settings 1 Expand the target experiment in the Experiment Explorer and double click the component you want to configure A public 0 A usert 24 a BD Experiment 12 26 2013 4 26 01 PM amp Experiment Information 4 E Sample Group 1 amp Sample Group Information Compensation Settings A Compensation Panel 4 Tube 1 1 Tube Information The corresponding dialog for the selected component appears Specify each parameter then click Apply or OK Example If Sample Group Information is selected Sample Group la Sample Group Information Sample Group name Sample Group 1 Date 12 19 2013 11 39 19 AM Species Cell type Memo OK Cance Apply For details about items displayed in the dialogs see Experiment Explorer page 133 For details about Measurement Settings see Configuring an Experiment page 73 Exporting Importing an Experiment You can import and export experiment data to back up the data to reopen a previous experiment or to review the experiment on another SH800 system Exporti
62. and Sheath Line page 162 As required Releasing Air in the Sheath Filter and Sorting Chip page 181 e As required Releasing Air in the DI Water Filter page 182 Releasing air trapped in the sheath filter and sorting chip Releasing air trapped in the DI water filter Autoclaving the sheath filter and DI water filter e As required Autoclaving the Sheath Filter and DI Water Filter page 183 e As required Ejecting the Sorting Chip Manually page 185 e As required Cleaning and Handling of Optical Filters page 186 Ejecting the sorting chip manually Cleaning the optical filters Disconnecting As required Disconnecting and reconnecting and Reconnecting the fluidics cart the Fluidics Cart page 187 Cleaning the internal sheath and DI water line with ethanol e As required Cleaning the Internal Sheath Line and DI Water Line using Ethanol page 188 Fluidics Cart Status of fluidics cart tank The status of the three fluidics tanks in the fluidics cart is displayed on the LCD monitor in addition to the Hardware Status pane in the bottom right corner of the main window in SH800 Software Sheath Pressure 20 00 psi Sample Pressure 20 70 psi Sheath Ethanol Fluidics cart lock mechanism The fluidics cart is fitted with a simple lock to prevent the tray from being withdrawn during operation The cart should only be unlocked when performin
63. and the bandwidth of the optical filter e g 525 50 for 525 nm bandpass optical filter 50 nm bandwidth The FSC and BSC bandpass optical filter labels have an F and B suffix respectively e g 488 17F for 488 nm bandpass optical filter with 17 nm bandwidth for forward scatter detection e Optical filters not used in the current filter pattern can be stored in the RESERVE 1 to 3 slots Optical Filter Pattern 1 Noa LP4 Optical Filter Pattern 2 BSC 488 17B Fluorescent Protein Optical Filter Pattern Option Filter Pattern for BV PE PI Optical Filter Patterns sno ueLjj s g xipueddy 203 sno ueLjj s g xipueddy 204 Fluorochrome Detection Matrix 1 laser 488 nm optical filter pattern 1 Fluorochrome Channel semok a 488 nm laser EGFP Enhanced GFP FL1 525 50 525 50 co mm oe PE RPhcenny ar 617 30 Perc nn bah pony ars poss O e 2060 ess 0a Aae aA Ae 617 30 AMN AMAN AN AN Ae Fluorochrome Detection Matrix 2 lasers 488 and 638 nm lasers optical filter pattern 1 FLI 52550 FLT 52550 7 AAD 7 Aminoactinomycin D FL5 72060 FL5 720 60 APC Alexa Fluor 750 FL6 785 60 Fluorochrome Detection Matrix sno ueLjj v s y g xlpueddy 205 snosuel aosiy g xipueddy 206 2 lasers 405 and 488 nm lasers
64. appear randomly and can occur in close proximity within the same droplet or in adjacent droplets called coincidences When a cell is detected during sorting it is generally not possible to know with great accuracy if the signal represents the passage of a large single cell small multiple cells adhering to one another or other non cellular material Accordingly it is more common to refer to each signal detection not as a cell but as an event The sorting mode sets the rules used when sorting coincidences These rules determine the sorting decision for each event based on the type of event and the distance between adjacent events For example a decision might be made to sort an event regardless of an adjacent unwanted event if the goal is to isolate as many cells as possible from a small population Conversely a decision might be made to not sort an event if there is an adjacent unwanted event that could reduce the purity of the sorted population Tip The trigger threshold setting in the Detector amp Threshold Settings dialog page 79 can be critical for effective sorting If the threshold level is set too high small cells and cell debris noise that fall below the threshold value may be unintentionally sorted together with events above the trigger threshold depending on the sorting mode setting To increase sorting purity it 1s recommended that the trigger threshold value be set to a relatively low value
65. appears 3 Click OK A Click Close to close the Backup Database dialog Restoring a Database Tip Restoring a database loads the information from a previous database backup erasing and replacing all information in the current database 1 Click Database on the File tab of the ribbon then click Restore Ba Delete All Data WW 6 ar the database Administrator Only The Restore Database dialog appears 2 Click Browse to specify the backup data to restore then click Restore Restore Database A Are you sure you want to restore the database Restoring the database will delete the current database and deleted data cannot be restored utdown upon selecting restore data Elapsed Time 00 00 00 A confirmation message appears 3 Click Yes SH800 Software launches and the database restore operation starts When the database is restored a confirmation message appears 4 Click OK 5 Click Close to close the Restore Database dialog Backing Up Restoring the Database 69 uoneiedo oiseg g13 deyo Kii s yu wn dx Buunbyuog p Je deyD Configuring Experiments Chapter This chapter describes how to customize experiments and how to configure experiment components detector settings and fluorescence compensation For details creating an experiment quickly see Creating an Experiment page 47 Creating a Customized Experiment You can cust
66. are displayed when right clicking a sample group icon in the Experiment Explorer New Tube Creates a sample group Selecting this item creates anew sample group for the experiment Copy Paste Allows you to copy and paste all settings including sample tubes in the selected sample group However the recorded data of the sample tubes will not be pasted Paste to All Tubes in Sample Group Pastes copied worksheet settings to all sample tubes in the selected sample group This item is available when you copy the worksheet settings of a sample group and then right click another sample group Duplicate Duplicates the selected sample group Save as Template Displays the Save as Template dialog page 115 for saving the selected sample group as a private template Apply Template Displays the Apply Template dialog page 115 for selecting a template to apply to the selected sample group Export FCS file Displays the FCS File Export page 117 dialog for exporting the recorded data for the sample tubes included in the sample group as an FCS 3 0 or FCS 3 1 format file Delete Deletes the selected sample group Rename Renames the selected sample group Measurement settings The following menu commands are displayed when right clicking a Measurement Settings icon for a sample group in the Experiment Explorer Copy Paste Copies the measurement settings for the selected sample group and paste
67. as an example 1 Grasp the short length of tubing with one hand and push the quick release collet into the connector using your other hand 2 Pullthe tubing out of the connector Changing Tank Air Filters soueuaulel g seideyuD 165 soueuajUleW g sajdeyuy 166 3 Insert the replacement air filter tubing into the connector and push firmly into place The filter may become clogged if fluid gets inside the filter adversely affecting unit operation If any fluid enters the filter while emptying replacing or refilling tanks replace the filter as soon as possible Cleaning the DI Water Tank and DI Water Line Cleaning the DI Water Tank and DI Water Line The DI water tank and line should be cleaned regularly with ethanol to suppress the growth of bacteria To suppress the growth of bacteria use sterile distilled water or sterile purified water as the DI water supply for normal use and cleaning Precautions when cleaning with ethanol e Ensure the laboratory is well ventilated during cleaning with ethanol Inhaling ethanol vapor can cause irritation of the eyes skin and respiratory tract loss of coordination drowsiness and in sufficient concentration unconsciousness e Never place a naked flame embers or other material that can emit sparks near the main unit or the fluidics cart Ethanol vapor is highly flammable at normal room temperature e Do not place any fluid other than the specified fluids
68. as described in To release air trapped in the sheath filter manually page 181 13 Click Ready in the Pressure Options tab of the Advanced Settings dialog The sheath fluid flow restarts 14 Load and run a sample tube containing sheath fluid and check for leaks around the connectors Check the drip tray for any fluid spills or leakages and clean as necessary Changing the DI Water Filter The DI water filter in the fluidics module should be replaced regularly to prevent small particles of dust that can cause fluctuations in the fluid flow and bacteria that can cause contamination of the sample DI water filter With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel 2 Open the fluidics maintenance door and remove the DI water filter from the retaining clamp without disconnecting the connectors Changing the DI Water Filter soueUuslUle y g Jaideyy I 177 suLeuguIeNw g Ja deuy 178 3 Disconnect the DI water filter lines from the UPPER and LOWER connectors Press the metal release catch and withdraw each connector The DI water filter connectors have stop valves that prevent leakage of fluid when disconnected Discard the used DI water filter Discard the DI water filter responsibly in accordance with local and federal ordinances and regulations Hold a new DI water filter with the arrow on the side o
69. be highly contagious or toxic depending on the sample under test Always wear gloves and other protective clothing mask and goggles as required when handling waste fluid With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel of the main unit 2 Pull out the fluidics cart tray 3 Unscrew the cap on the top of the waste tank and remove the waste tank nozzle Emptying Changing the Waste Tank 4 Place the cap with nozzle connector and line attached in a beaker or other container to prevent waste fluid spills from the nozzle e Do not touch the surfaces of the waste tank cap and tank nozzle that come into contact with waste fluid Take care not to spill any fluid e Place the beaker in a safe position where it cannot be accidentally moved or tipped over 5 Attach the inner cap on the tank taking care not spill any waste fluid then attach the outer cap and securely tighten The inner and outer stopper caps are supplied with the tank Always attach both the inner and outer caps to ensure the tank is sealed 6 Remove the waste tank from the fluidics cart tray 7 Empty the tank or prepare a new waste tank Dispose of the waste tank and fluid in accordance with the laboratory rules and local ordinances and regulations 8 Place an empty waste tank or a new tank on the fluidics cart tray 9 Insert the waste tank nozzle from the beaker into the tank secure
70. contact your Sony distributor for service Check that there is no object shielding the base of the cytometer If the problem persists contact your Sony distributor for service The cytometer may be overheating Shut down the cytometer and allow the temperature to fall within the rated range before rebooting If the problem persists at temperatures not exceeding 35 C 95 F contact your Sony distributor for service Error Messages Ssnosuel aosi g xlpueddy 219 sno ueLjj s g xipueddy 220 Shut down the cytometer and wait a while before trying ifthe problem persists contact your Sony distributor for Temperature error from the X axis in the chip stage motor on the MD2 board Temperature error from the Z axis in the chip stage motor on the MD2 board Temperature error from the chip load motor on the MD2 board Temperature error from the collection load motor on the MD2 board Temperature error from the agitation motor on the MD3 board Pump error on the MD1 board Temperature error from the droplet camera motor on the MD2 The cytometer may be overheating Shut down the cytometer and allow the temperature to fall within the rated range before rebooting If the problem persists at temperatures not exceeding 35 C 95 F contact your Sony distributor for service Shut down the cytometer and wait a while before trying ifthe problem persists contact your Sony distributor for
71. depend on the wavelength of the lasers and the optical filter pattern The fluorochrome detection matrix for each laser configuration is given in the Fluorochrome Detection Matrix page 204 Tip On multi laser models you specify the lasers used for experiments when SH800 Software launches e Do not use the 405 nm laser for extended periods of time Extended use can damage the sorting chip e The use of optical adjustment procedures not specified in this document or installation removal of optical components may result in hazardous radiation exposure Do not remove or otherwise modify the excitation laser or detection module Series Lineup sno ueLjj v s y g xlpueddy 201 sno ugjj siy g X pu ddy 202 Optical Filter Patterns The detection module takes the scattered and fluorescent light collected from cells and separates it into eight channels of light comprising the forward scatter channel back scatter channel and six channels of fluorescent light of various wavelengths The SH800 uses a combination of longpass optical filters in slots labeled LP1 to LP5 and bandpass optical filters in slots labeled BSC FSC and FL1 to FL6 arranged in an optically optimized layout according to the laser configuration for an experiment The SH800 uses two standard optical filter patterns Optical filters Optical filter Optical filter coins patent pate 1 On the following models dummy filters are
72. different temperature to the sheath fluid remaining in the tank It is recommended that the sheath tank be filled one day before measurement to ensure all air bubbles are removed from the sheath fluid 1 Pull out the fluidics cart tray 2 Disconnect the sheath air line clear from the top of the sheath tank Refilling the Sheath Tank 4 It is important to disconnect the sheath air line before disconnecting the sheath fluid line Release the residual air pressure from the tank using the ring pull air relief valve then remove the tank from the tray Disconnect the sheath fluid line blue from the top of the tank 5 Lift the lever securing the lid of the tank to open the lid Fill with sheath fluid to below this line 8 Insert and secure the lid place the tank in the cart tray and connect the sheath air line clear and sheath fluid line blue in the reverse order of removal making sure the connectors are inserted until they lock into place 6 Lower the lid slightly into the tank turn it 90 EEJ Eaa degrees and then remove the lid from the tank When inserting the sheath tank lid make sure that the black rubber seal is attached to the lid 4 4 9 Carefully clean any residue fluid from the connectors and outside surfaces of the tank and stow the tray Always place the tank in the center of the tray area AS The fluid level detector may not operate correctly 1f a the tank is placed off center
73. do not match the sort criteria and which are aborted during sorting Abort Rate Displays the rate at which events are aborted during sorting in units of events per second eps Droplet pane Displays the droplet stream immediately beneath the sorting chip nozzle The droplet stream is used to monitor droplet formation and to adjust the breakoff point Double clicking the image displays the side streams in the Droplet Viewer window page 138 for monitoring sorting Hardware Status pane Displays the status of the interior lights in the sample loader and collection stage area and the level in each of the tanks in the fluidics cart It also displays the lasers enabled for the experiment Light Collection Turns the collection area light on off Sample Turns the sample chamber light on off Tank Indicates the fluid level in the sheath tank ethanol tank waste tank and DI water tank A warning icon appears when the waste tank is close to full or when the sheath ethanol or DI water tank is close to empty Laser Indicates the lasers used for data acquisition For 8 well strips or slide glass sorting The following items are displayed when 8 Well Strips or Slides is selected in Sorting Method The Droplet and Hardware Status panes are the same as for two way tube sorting Sort Control pane Configures and controls droplet sorting Sort Start Sort Stop Starts stops sor
74. during sorting Do not attempt to remove a multi well plate while a Stop If data acquisition is in progress click the Stop to stop data acquisition recording or M the collection stage 1s moving Select the appropriate plate sorting method in Pause to pause data acquisition recording iy Sample Group 1 Jy E Tube 1 Status Pause f Elapsed Time Total Event Event Rate 0 eps gt Restart Start Stop Record hiii j j Sample Stop Condition None Y 2 Attach the splash guard Always attach the splash guard when sorting into multi well plates For details see Attaching the Splash Guard for use with multi well plates SHSOOZP only page 35 3 Place the 96 well plate into the corresponding holder For details see Preparing Collection Tubes page 33 4 Open the sorting area door place the holder on the collection stage and close the door 96 well plate holder Sorting Sorting Method Always select the sorting method matching the collection tube device loaded on the collection stage ka Dort Control a a Load Collection fae Sort Settings The sorting parameters vary depending on the selected sorting method Click Sort Settings Ww Sort Control Load Collection Sortmg Method 96 Well Plate
75. for data acquisition recording and sorting for the tube Double clicking this component displays the Stop amp Sort Settings dialog page 135 A Data source Indicates the presence of saved data and type of sort results Data source naming convention The sorting method is prefixed to the name of the data source 6 Well Plate 6 Well Data Source 12 Well Plate 12 Well Data Source 24 Well Plate 24 Well Data Source 48 Well Plate 48 Well Data Source 96 Well Plate 96 Well Data Source 384 Well Plate 384 Well Data Source Main Window Index sorting Index 96 Well Data Example 96 Well Source Plate The data source name has an Index prefix Imported data created Old 8 Well Data Source using SH800 Software version 1 3 1 or earlierEx 8 Well Strips Old prefix The data source name has an Experiment Information dialog The Experiment Information dialog is displayed by double clicking Experiment Information for an experiment in the Experiment Explorer The Experiment Information dialog is used to view and edit information about the experiment Experiment 12 28 2013 9 45 52 PM Experiment Information Experiment name Experiment 12 28 2013 9 45 52 PM Experiment Name Name of the experiment required item Date Displays the date the experiment was created This parameter cannot be modified Investigator Name of the lead research
76. in the DI water tank e Make sure that the cap on the waste tank is correctly and securely fastened at all times The SH800 is compatible with standard disinfecting agents ethanol 70 used for flow cytometers With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel 2 Open the fluidics maintenance door then disconnect the line from the top of the DI water tank Press the metal release catch to withdraw the connector 3 Undo the metal restraining clamp and remove the DI water tank Always wear gloves and other protective clothing mask and goggles when handling fluidics system components Skin cells and hair are the most common cause of bacterial contaminations Remove the cap and withdraw the tank probe Disconnect the quick release connector from the air filter Discard any remaining DI water Dispose of DI water in accordance with laboratory rules and with local and federal ordinances and regulations Pour 0 5 liters 0 15 gallons US of ethanol into the DI water tank and reattach the cap Shake the tank while holding your finger over the air filter connection port to clean the tank Cleaning the DI Water Tank and DI Water Line soueUuslUle g Jaydeyy I 167 eoueuajUleyy g Jajdeyy 168 9 Remove the DI water filter and connect the DI water filter bypass line green Shake gently while holding here 1 0 Turn on the powe
77. is in one half of the droplet the nearest adjacent droplet is also sorted if the nearest half of the subsequent droplet is empty or non conflicting One or two droplets are sorted for each event matching the sorting criteria Sorted Target cell is in main droplet and adjacent droplet and nearest half of subsequent droplet are empty Not sorted Conflicting cell is in nearest half of adjacent droplet __ _ 4 Not sorted Conflicting cell in nearest half of subsequent droplet above _ __ lt r Not sorted Conflicting cell is in nearest half of adjacent droplet Ultra Purity Mode In Ultra Purity mode cells are sorted with the highest purity 100 um sorting chip A droplet is sorted if it contains one or more targeted events of a single type and the both of the adjacent droplets are empty or non conflicting One droplet is sorted for each event matching the sorting ar JE S lt Not sorted Conflicting cell is in adjacent droplet 130 um sorting chip A droplet is sorted if it contains one or more targeted events of a single type and the both of the adjacent droplets are empty or non conflicting In addition the adjacent droplet is also sorted 1f the subsequent droplet is empty or non conflicting One two or three droplets are sorted for each event matching the sorting criteria VQ Q Not sorted Conflicting cell in the subsequent droplet above lt t Not sorte
78. line compatible Non PEEK sample line compatible Model Identification Components and Documentation Components and Documentation System Components The SH800 system comprises the following items e Main unit 1 e Fluidics cart 1 Safety Guide 1 Operator s Guide this document 1 Installation disc Operator s Guide software 1 Power cord 1 e PC connection cable 1 PEEK sample line 1 and probe adapter 1 combination PEEK sample line compatible models Sample line 1 and sample probe 1 combination non PEEK sample line compatible models Sheath filter 1 Sheath filter bypass line yellow 1 DI water filter bypass line green 1 DI water filter 1 Drip tray 1 DI water tank 1 DI water tank probe 1 Air filter for DI water tank 1 Sheath tank 1 Ethanol tank 1 Ethanol tank probe 1 Air filter for ethanol tank 1 Waste tank 1 e Waste tank nozzle 1 Air filter for waste tank 1 Sheath air line clear for fluidics cart 1 Sheath line blue for fluidics cart 1 Ethanol line yellow for fluidics cart 1 Waste line red for fluidics cart 1 e Cart connection cable 1 Sample tube holder 0 5 ml 1 Sample tube holder 1 5 ml 1 Sample tube holder 5 ml 1 Sample tube holder 15 ml 1 Cleaning tube holder 30 ml 1 Collection tube holder 5 ml 1 Collection tube holder 15 ml 1 SH800ZP standard equipment e 96 well plate hold
79. making it difficult to discern patterns In this case reduce the number of events displayed or use a density plot to view the data Editing Plots 1 000 sgg 600 BSC A 400 200 41 000 Histogram plots Histogram plots display a frequency distribution of fluorescence intensity for a single acquisition parameter univariate distribution Histogram plots are effective for identifying the peaks and troughs in measurement data All Events 1 000 Events B25 11 000 475 650 FSC A 125 300 Changing Plot Type Select a plot whose type you want to change on the worksheet Tube 1 Data Source 1 Tube 2 Data Source 1 gt All Events 2 Click the target plot type in the Plot Type group on the Plot Tools tab of the ribbon You can also right click a plot and select a plot type from the Plot Type submenu of the context menu For example the diagram below shows a density plot being changed to a histogram plot eee Worksheet Tools sriment Cytometer Compensation Tube 2 Index 96 Well Data S E A a Density Xt Fa 7 X Axis Linear X m lz ee Dot Plot R my New Remove Duplicate L_ Y Axis Linear v Swap Histogram Piot Piot lads Histogram rig The type of the selected plot is changed All Events Duplicating a Plot Selecta plot to be duplicated on the worksheet then click Duplicate Plot in the Edit group on the Plot Tools
80. matrix for the sample group For details see Performing Fluorescence Compensation page 61 Show Matrix Displays the Compensation Settings dialog page 124 for viewing the spillover matrix and loading saving the spillover matrix Calculate Matrix Displays the Calculate Compensation Settings dialog page 125 for calculating the coefficients of the fluorescence compensation spillover matrix based on the negative control tubes and the compensation panel Apply compensation Selects whether fluorescence compensation is applied to plots on the worksheet Clicking this button toggles compensation on off Manual Compensation Toggles manual fluorescence compensation mode on off for the plots on the worksheet Compensation Settings dialog The Compensation Settings dialog is displayed by clicking Show Matrix on the Compensation tab of the ribbon The Compensation Settings dialog is used to view the spillover matrix and load save the spillover matrix Sample Group 1 Compensation Settings Spillover Matrix Detector Brilliant Vio u Brilliant Vio 0 00 0 00 0 00 s PerCP Cy5 5 PE Cy7 Oo co oS 0 00 0 00 0 00 Fluorochrom O uv m 0 00 gt 0 00 0 00 0 00 APC 0 00 0 00 0 0 00 0 00 PerCP Cy5 5 0 00 0 00 0 0 00 0 00 PE Cy7 0 00 0 00 0 0 00 0 00 Al Negative Value jrilliant Vie Perl P y5 5 f f f Ares 0 0 0 Heig ht 0 0 0 Tip Mat
81. on the LCD monitor Precautions when handling ethanol Ensure the laboratory is well ventilated during cleaning with ethanol Inhaling ethanol vapor can cause irritation of the eyes skin and respiratory tract loss of coordination drowsiness and in sufficient concentration unconsciousness e Never place a naked flame embers or other material that can emit sparks near the main unit or the fluidics cart Ethanol vapor is highly flammable at normal room temperature e Never pour ethanol into a tank other than the ethanol tank and conversely never pour other liquids into the ethanol tank e Make sure that the cap on the ethanol tank is correctly and securely fastened at all times e Make sure that the ethanol line is securely connected to the connectors on the rear panel of the main unit rear panel of the fluidics cart and the ethanol tank itself e When the ethanol tank is not in use remove all ethanol from the tank The SH800 is compatible with standard disinfectant ethanol 70 intended for use in flow cytometers 1 With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel of the main unit 2 Pull out the fluidics cart tray 3 Disconnect the ethanol line from the cap on the ethanol tank and place it in a clean beaker or other container Refilling the Ethanol Tank soueuslUle g Jaideyy I 151 soueuajUley g sajdeyy 152 6 7 Press the metal r
82. panel are recorded If the sensor gain of any detector is changed after recording the control tubes the fluorescence compensation will no longer be valid and the control tubes will need to be loaded and recorded again 1 Click Compensation Wizard on the Compensation tab of the ribbon me Al Compensation Cytometer Experiment Be Show Cabculate Mtatrix Matrik Compensation Manua Wizard The Compensation Wizard launches 2 Click Next Compensation Wizard lt lt The compensation wizard will guide you through the steps of creating a spillover matrix Please prepare negative control tube and each single stained tubes and click Next button The following screen appears Follow the on screen instructions to record control tubes The operating procedure is displayed Compensation Wizard lt lt Load the negative control tube indicated and select Start button to acquire the sample 1 Gain Settings A control tube is automatically assigned as the active tube and the worksheet for the tube opens 3 Load the control tube displayed in the wizard then click Start on the data acquisition control pane Status Ready 00 00 00 Total Event 0 FHlapsed Time Event Rate 0 eps SE Tip If for any reason you choose not to use a fluorochrome channel you can click Skip to bypass the control tube Compensation Wizard lt lt Load the single color control tub
83. parts to sterilize the filters e Only the filters are autoclavable e Do not autoclave the filters more than once at a time e Each filter can be autoclaved up to ten times Before autoclaving Check that the filter has a CER marking autoclavable parts The filter marking 1s on the bottom of the filter body Check the label CER Autoclavable CSS Non autoclavable 1 Remove the filter from the main unit For details about removing filters see Changing the Sheath Filter page 176 and Changing the DI Water Filter page 177 2 Unscrew the IN and OUT connectors from the plastic couplers and remove the cap and filter spacer from the filter Autoclaving the Sheath Filter and DI Water Filter soueUuslUle 9g Jaideyy IN 183 eoueuajUleyy g Jajdeyy 184 Do not remove the metallic couplers Go Dispose of any fluid inside the filter Drain the fluid from the filter until there is no remaining fluid 4 Autoclave the filter under the following conditions Temperature 121 C 250 F Pressure 2 atmospheres Duration 30 minutes 5 After autoclaving attach the couplers and reinsert into the main unit G Attach the IN and OUT connectors Tightening torque 0 50 0 05 N m If the cap has a washer check that the washer is inserted correctly for proper sealing of the cap Autoclaving the Sheath Filter and DI Water Filter Correct O Inserted straight Incorrect
84. plate holder 384 well PCR adapter 1 Options Common to all models e Collection tube holder 15 ml Vi a e Collection tube holder 5 ml Vi T LA When running 2 way tube sorting on the SHS00ZP the plate holder adapter must first be removed See Preparing Collection Tubes 33 uonesedalg gieideyy HIN uoneledaig zguisajdeyy Removing the Plate Holder Adapter SHSOOZP e 96 well PCR plate holder with 96 well PCR plate only page 37 SH800 and SH800Z e 8 well strips holder with 8 well strips e 384 well plate holder with 384 well plate For details about loading 8 well strips see Loading S well Strips SH800 and SHS800Z on page 35 e Slide glass holder For details about loading tubes see Loading a Multi well Plate SHSOOZP only page 36 e 384 well plate holder with 384 well PCR adapter and 384 well PCR plate SH800ZP only e 96 well plate holder with 96 well plate For details about loading tubes see Loading a 384 well PCR Plate page 36 When sorting into a 384 well plate or 384 well PCR plate always adjust the sort position before starting For details see Adjusting the Sort Position SHSOOZP only page 99 34 Preparing Collection Tubes DANGER Attaching the Splash Guard for Fluids may contain biological chemical or other agents use with multi well plate S When handling collection tubes always wear
85. right hand side shows the current event rate relative to the guaranteed performance rate of 10 000 eps 5k 10k 20k Tip It is recommended that the event range be reduced if the indicator exceeds a rate of 10k Data acquisition controls Start Starts resumes data acquisition It does not start data recording Pause Pauses data acquisition The elapsed time continues counting while acquisition is paused Stop Stops data acquisition manually lt Record Records the acquired data REC stop Stops data recording manually Restart Resets the total events and elapsed time counters to 0 and restarts data acquisition Tip If an automatic stop condition has been specified data acquisition and or recording stops automatically when the corresponding stop condition has been satisfied Sample Stop Condition Selects the stop condition for stopping data acquisition automatically e None data acquisition will stop automatically when the number of detected events exceeds 100 000 000 or the elapsed time exceeds 12 00 00 whichever occurs first e Recording stops data acquisition automatically when the recording stop condition is satisfied e Sorting stops data acquisition automatically when the sorting stop condition is satisfied e Recording and Sorting stops data acquisition automatically when both the recording and sorting stop conditions are satisfied Instrument controls Load Sample Unload
86. sample line to the sample probe then click Next Hold part B of the sample probe and screw part C on the sample line until it clicks into place making sure it is securely attached Part C Part B If reattaching the removed sample line and sample probe disinfect the components using the following procedure as required e Place the sample line in a beaker or other container of ethanol and let sit for about 30 minutes e Autoclave the sample probe under general autoclave conditions 121 C 250 F 2 atmospheres approx 20 minutes Insert the sample probe while pressing its metal release catch to the right Press down on the sample probe to insert it securely and release the metal release catch Verify that the metal release catch has returned to the left side 9 Open the flip up door and attach the new sample line to the inlet port on the front of the chip loader then click Next Screw part A until it clicks into place making sure it is securely attached soueuslUle g Jaydeyy I Changing the Sample Line and Probe Non PEEK sample line compatible models 173 eoueuajUleyy g Jajdeyy 174 When attaching the sample line always hold part A of the connector not the T shaped portion Holding the connector by the T shaped portion may cause tightening that exceeds the appropriate tightening torque and adversely affect instrument performance e When attaching the sample line connector alway
87. sample to increase the concentration of the cells or to isolate cells for increased purity Supported sorting methods for sorting chip nozzle diameter The sorting methods that can be used vary depending on the nozzle diameter of the sorting chip and the size of cells SH800 and SH800Z Sorting method 2 Way Tubes 8 Well Strips Slides Cell size settings Regular Cell Large Cell Regular Cell Large Cell Regular Cell Large Cell SH800ZP om ves e Sorting into 8 well strips multi well plates or onto glass slides 1s not supported when using the 130 um sorting chip e Large Cell is an assistive mode for the sorting of large sized cells when using the 100 um sorting chip Use it if you are having trouble sorting large cells However this function does not guarantee complete accuracy in sorting large cells A rough guide for the size of large cells for which to use the Large Cell setting is 15 um or larger 3 Open the sorting area door place the holder on the collection stage and close the door Two way Tube Sorting Check that data acquisition and recording are SH800 stopped or paused If data acquisition is in progress click the Stop to stop data acquisition recording or Pause to pause data acquisition recording BI Sample Group 1 i E Tube 1 Status Pause Elapsed Time Total Event Event Rate 0 eps wa gt j Restart Start Stop Record Sample Sto
88. sample tube holder containing a single sample tube containing sample fluid into the sample loader agitation unit Samples can be loaded in 0 5 ml or 1 5 ml micro tubes 5 ml round tubes or 15 ml conical tubes A sample tube holder is provided for each size of sample tube For details see Loading a Sample Tube in a Sample Tube Holder page 31 Internal View Side al 1 Fluidics maintenance door Sheath filter DI water tank Drip tray Fluidics maintenance door Provides access to the fluidics system for performing maintenance Be aware of the door edges when the door is open Sheath filter Filters the sheath fluid flowing between the sheath tank and the sorting chip The sheath filter is a maintenance item DI water tank Supplies deionized DI water used for flushing the fluidics system A sensor detects the level of DI water within the tank and a message 1s displayed when the level drops below the minimum threshold The tank has a capacity of 1 liter 0 3 gallons US The DI water tank is a maintenance item Drip tray Collects liquid spills during routine maintenance and leakages due to damaged tubing or improperly fitted connectors The drip tray should be checked regularly for signs of leakage An alert is shown on the host computer if fluid is detected in the drip tray If fluid is detected resolve the source of the leak clean the drip tray and wipe clean a
89. samples in the experiment from the main window Recorded data is displayed in real time and can also be exported for analysis at a later time Before starting acquisition Check the following items e Check the fluid levels in each of the tanks e Check that the main unit and fluidics cart are connected correctly You control data acquisition and recording in the Acquisition tab of the Experiment Acquisition control Copy Show Gates and Statistics Events Parent Total Sort ficiency Acquisition tab Insert sample fluid in a sample tube and place the tube in the corresponding sample tube holder For details about sample tubes and sample tube holders see Preparing a Sample page 31 2 Place the sample tube holder in the sample loader If the sample loader door is closed press the sample loader door button to open the sample loader door 3 Press the sample loader door button to close the sample loader door Tip The sample loader door will close automatically 1f you leave the door open when you click Start in SH800 Software to start acquisition 4 Specify the measurement stop condition in Sample Stop Condition Total Event Event Rate 0 eps u Restart Start Stop Record Sample Stop Condition None 7 i Unload Sample Detector amp Threshold Settings Sample Pressure 4 X The following options are available e Non
90. sheath fluid in the tank 1 0 Place the tank in the cart tray and connect the sheath 11 Dispose of the sheath fluid in accordance with the laboratory rules and local ordinances and regulations Pour 3 liters 0 8 gallons US of ethanol into the sheath tank and reattach the lid e Do not touch the inner surface of the lid of the sheath tank Also take care not to spill any ethanol e Do not use a pump or other object that has not been sterilized for pouring ethanol into the tank e When inserting the sheath tank lid make sure that the black rubber seal is attached to the lid Grasp the handles and shake the sheath tank to clean the inside of the tank if necessary Grasp the handles and shake the tank jel 2 Sole fluid line blue and sheath air line clear in the reverse order of removal until they lock into place Always connect the sheath fluid line blue and sheath air line clear in that order Connecting the sheath air line first will pressurize the sheath tank causing ethanol to seep out of the sheath fluid line Clean any fluid from the connectors and outside surfaces of the tank and stow the tray Insert the line connector and push until it locks into place Clean any fluid from the connector and outside surfaces of the tank Cleaning the Sheath Tank and Sheath Line soueuaule g seideyo I 163 soueuajUley g Jajdeyy 164 1 2 Disconnect the sheath filter lines in th
91. that the surface of the chip is not dirty or smudged then perform chip realignment If the sorting chip is dirty or smudged replace the chip The sorting parameters may be set incorrectly The sorting parameters affect the droplet shape breakoff point and electric charge Increase the amplitude or decrease the frequency to make droplets larger Decrease the amplitude or increase the frequency to make droplets smaller Sheath fluid is not flowing Check that the connectors for all fluidics lines in the fluidics cart and main unit are connected securely and that the lid of the sheath tank is closed tightly Check that the sheath tank lid has a black rubber seal attached Check that there are no air bubbles in the sheath filter Check the pressure of the compressed air supply to the main unit sno ueLjj s g xlpueddy Electrical discharge between deflection Discharge may occur due to fluid droplets adhering to the surface of the plates plates causes sorting errors Turn off the power open the sorting area door and wipe the deflection plates dry If residue remains on the plates remove and clean the plates PMT output signal does not change after Increasing the gain too much can cause output saturation In this case reduce increasing the gain the gain and readjust settings to find the optimum operating point Sorting chip does not load correctly Log off and restart in maintenance mode Running Maintenance Mode page 158
92. the droplets is determined by the nozzle size frequency of vibration and the speed of the fluid flow The amplitude of vibration drop drive voltage controls the position of the breakoff point and the frequency of vibration determines the distance between droplets If the last attached droplet satisfies the sorting criteria the droplet containing the cell receives an electrostatic charge just before the fluid flow reaches the breakoff point from a charging electrode located near the sheath fluid inlet on the sorting chip The last attached droplet retains the electrical charge after it breaks off from the fluid flow The electrostatic charge is distributed on the periphery of the droplet and does not affect any sample cells contained within the droplet Fluid Flow The polarity positive or negative of the charge applied to a droplet depends on the cell sorting criteria defined by the characteristics of the cell populations that you want to isolate for sorting using gates specified in SH800 Software This information is then used to drive the charging pulse for sorting Unwanted droplets are left uncharged and are collected by the waste catcher In an ideal sort all cells travel pass the optical detection point in single file and are sorted one cell at a time in separate droplets with an interval between droplets In practice however cells can appear very close together at the optical detection point which means they may be foun
93. the Axis Parameters cccccccceccceeceeeeeeeeeeeeeeeeenaas 86 Adjustine Scale Ranges inrer a 86 Adding Gales consist Stee 88 Adin Sa Physical Gate resisa 88 Copy me a Physical Grate menaire ister dteeeten dees 89 Adding a Boolean gate to the Gate Hierarchy eee 89 Editing Gale asincrona aaa aia 91 Displaying Statistics ccccsseesesceceseeeeseseneesenseseneeeeseeoees 92 Chapter 6 Sorting SONING MOUE sinaia aae aR 93 NOINE WV a N setaacsaitananeaenmnestetban vent 93 Ingle Ce UMO dE apran a entails 95 Norimak Mode cuecen etaa A E 96 DEM Pury Mode sc sepssniades deste 96 Purity Mode ereny en E ie ateaenaeiuated 97 Ultra POr ModE eero a ter Taree mere rrrr werner Serr er 97 SEMEN 161 GIVI OC sccssesssaasituanacheatee serwaseunded ou taaduadpsasahaataetawaeanlte 98 AV MCMC INIOUG anansse ie alsa saath ae ticiatieeie eisai tae ates tebe iain abed 98 Uira Y cld MOda enone amsaneiuiaasaaee 99 Adjusting the Sort Position SH800ZP only 99 Nde SOUNO soruiri a EEEE 101 Configuring Index Sorting oseessssssseseeressssssseeerrssssssseeeeees 101 Analyzing Index Sort Data ssciitossonaicrionnopeesaonk 102 Adjusting Sort Parameters Manually Experienced Users cccccsscsesscseseeesseesseesseeeeeeneees 103 Chapter 7 Window Description Table of Contents UE VV GOW ieni a 104 nlora Non WiINdOW aissa E ets 104 PPG Wi OW a a 108 Pata ase WMO We mera r ate uale dest iteee
94. the gain of a detector for any channel is changed for any reason after recording the control tubes the fluorescence compensation will no longer be valid and the control tubes will need to be loaded and recorded again For details about recording control tubes in a compensation panel see Performing Fluorescence Compensation page 61 Tip There are various other methods that can be used to optimize the detector gain settings for an experiment For example a more rigorous approach to detector gain optimization might be to measure the coefficient of variation CV for positive controls at various detector gain levels to determine the minimum gain levels for which the CV plateaus off in each detection channel sensor A detector gain set at this minimum gain level will provide the optimal signal to noise ratio required for maximum sensitivity and maximum separation between negative and positive peaks Adjusting Fluorescence Compensation Adjusting Fluorescence Compensation Using a Target Gate in the Gate Hierarchy You can specify the data for a gate within the gate hierarchy to be used as the negative sample data or a singly stained positive sample data by setting the gate as the target for calculating the compensation matrix 1 Right click a gate on a plot for a control tube in the compensation panel to set the gate as the calculation target for the compensation matrix and select Set As Calculation Target from the cont
95. to SSC Selects whether to change the sensor name for data recorded by the BSC back scatter detector to SSC side scatter when exporting FCS files Maximum Save Events Specifies the maximum number of events to save File Window uondos q mopuiM Z JeIdeyD I 107 uolduoseg mopuiM Z Je deuyD 108 Print Window The Print window is displayed by opening the worksheet of the tube you want to print and clicking Print in the menu on the left side The Print window can print analysis results statistics and plots displayed on the worksheet 00 9 Print button Sends the print job to the printer Print Sets the number of copies to print Place a check mark in the Use Printer Settings checkbox to set advanced printer settings Printer Selects the printer for printing Print preview area Displays a preview image of the print job Settings Sets the paper size and page orientation Database Window The Database window is displayed by clicking Database in the menu on the left side The Database window is used to perform experiment database operations and to check hard disk free capacity Example Administrator account login Import Displays the Import Experiment Data dialog page 109 for importing experiment data You can import experiment data created using another SH800 for use on this unit File Window Export Display
96. to modify 2 Click Apply Compensation on the Experiment tab of the ribbon The fluorescence compensation obtained using the Compensation Wizard is applied to the plots on the worksheet 3 Display the Compensation Settings dialog by doing one of the following e Select Compensation Settings in the Experiment Explorer and click Show Settings on the Experiment tab of the ribbon e Double click Compensation Settings in the Experiment Explorer e Select any component within the sample group and click Show Matrix on the Compensation tab of the ribbon The Compensation Settings dialog appears 4 Click a cell in the matrix to select its value You can adjust a value using one of the following methods e Turn the mouse scroll wheel button e Enter a value directly in the spillover matrix e Click the spin boxes e Drag the slider 82 Adjusting Fluorescence Compensation Sample Group 1 Compensation Settings Spillover Matrix Brilliant Vio t PE Cy7 S 8 PerCP Cy5 5 so 6 Ss v 5 Brilliant Vio 0 00 0 00 0 00 S FTC o 2 PE 0 00 000 SHF 0 00 gt o o o 5 o o oI 9 E APC 0 00 0 00 PerCP Cy5 5 0 00 0 00 0 0 00 0 00 PE Cy7 0 00 0 00 0 0 00 Negative Value Brillian Height The plots on the worksheet are updated in real time e The number of fluorochromes in the experiment determines the dimension of the spillover matrix Spillover elemen
97. turned off Sorting area door button Opens the sorting area door Sorting area door Provides access to the sorting and collection area for loading and unloading collection tubes for sorting It also provides access for cleaning and routine maintenance A safety interlock switch disables operation while the door is open A rubber packing seal is used to ensure the integrity of the sorting area door If the door does not open when the door button is pressed the door may be sticking to the packing In this case open the door by simultaneously Name and Function of Parts LCD monitor OL DISPLAY MODE button C POWER STANDBY button Cx Sample loader door button Sample loader door pressing the button with one hand while pulling gently on the bottom edge of the door with your other hand Sample loader door Provides access to the sample loader for loading and unloading samples for testing A safety interlock switch disables operation while the door is open Sample loader door button OPEN CLOSE Opens and closes the sample loader door Once a sample has been lifted into the injection chamber within the sample loader and acquisition started the button is disabled to prevent opening and closing of the sample loader door Observe the following precautions when opening and closing the sample loader door e Turn on the power before operating the sample loader door button e When opening a
98. weer wee es wes es es Oe _ tdTomato FL3 600 60 FL4 665 30 FL4 665 30 785 60 APC Alexa Fluor 750 ne FL6 785 60 Ae mPum APC Allophycocyanin ee em m OO oo x yo yN N ee ee N ee S SN Fluorochrome Detection Matrix sno ueLjj s g X pu ddy 211 sno ueLjj s g xipueddy 212 Relationship between Sample Pressure and Sample Flow Event Rate 100 um sorting chip ae ee Flow rate ul min 6 m 16 Event rate eps Sample 160 260 350 450 zs CEN aa concentration 1e6 ml Sample 1 300 2 100 2 800 3 600 5 100 6 600 8 100 9 600 12 600 concentration 1e7 ml 130 um sorting chip seem o o d a a e e a a Flow rate ul min Event rate eps Sample concentration 1e6 ml Sample 1 200 2 000 2 800 3 600 4 500 5 400 6 500 8 000 concentration 1e7 ml Relationship between Sample Pressure and Sample Flow Event Rate Connecting an AMS Evacuator SH800Z and SH800ZP You can connect a S A F E System evacuator from Edge Systems LLC using a specialized adapter AMS evacuator connection kit Compliance with US NIH National Institutes of Health biosafety policies is not guaranteed Rear of unit AMS evacuator For details on operating the evacuator and replacing filters refer to the operating instructions for the evacuator AMS evacuator connection kit components e HEPA filter bracket 1 e AMS tube joint
99. x 1080 16 9 Full HD Graphics resolution 1920 x 1080 Full HD Camera 1 3 megapixels or higher effective pixels OS language setting Symbol for the decimal point is 66 99 period 66 99 If the decimal point is set to a character other than period the following error message is displayed when starting SH800 Software Invalid Language setting was detected Please change Decimal symbol setting to period according to the Operator s Guide Press OK to close this application If this occurs use the following procedure to change the decimal point to period then restart SH800 Software 1 Open Control Panel gt Clock Language and Region gt Region and Language 2 Open Additional settings on the Formats tab 3 Set Decimal symbol to period on the Numbers tab SH800 Software Operating Environment 2 Gaa WN M Q Je deyD 28 LCD Monitor The LCD monitor on the front of the SH800 displays status information during operation The LCD monitor is turned on off using the Display Mode button on the front panel Sheath Pressure 20 00 psi Sample Pressure 20 70 psi Sheath Ethanol Waste DI 488nm Tip If conflicting information is displayed in SH800 Software display and on the LCD monitor it does not necessarily indicate a failure or error Sheath Pressure 99 99 psi Sample Pressure 99 99 psi Sheath Ethanol Waste DI 48
100. 100 100 300 100 100 10 100 100 100 10 100 100 G 100 100 5l OIC O o r 5 i PERA En sl e ol Close After one set has been sorted the following message appears SH800 Software Sorting is completed for all well Do you want to continue to next sort Sorting 5 7 uoneiedo oiseg g13 deyo HNN uoleisdo olseg Jsa deuyD 58 9 Click Finish to finish sorting To continue sorting click Continue When you click Continue the collection stage is unloaded allowing you to place the next set Sorting into Multi well Plates SH800ZP only This section describes the procedure for sorting into a 96 well plate The procedure is identical for sorting into 6 12 24 48 and 384 well plates Check that data acquisition and recording are stopped or paused If the multi well plate is loaded in the incorrect orientation correct sorting is not possible Check the orientation when loading a multi well plate Do not sort sample fluid above the permitted volume into the wells and do not overfill the wells with buffer solution to prevent sample fluid overflow from the wells Take care not to spill samples when removing the multi well plate from the multi well plate holder Securely mount the holder adapter to ensure successful sorting Always load a multi well plate of the same size as that selected in Sorting Method Do not open the sorting area door
101. 8nm LCD Monitor Preparation Chapter Checking Device Connections Check that the main unit fluidics cart host computer and compressed air supply are connected correctly Fluidics cart Cart connection cable Main unit r Sheath line blue tubing Ethanol line yellow tubing Waste line red tubing I Sheath air line clear tubing Air compressor or Main power switch compressed air supply MAIN POWER Power connection Notes e Make sure all fluidics line fittings are inserted into the corresponding connectors and are secured correctly There is a danger of fluid leakage 1f fittings are not properly sealed e Connect the power cord for the air compressor directly connection cable i to a power outlet Do not connect the power cord using Host computer p an extension cable Checking Device Connections 29 uonesedalg giaideyuy HIN uonweiedaid Z 1 deyo 30 Filling the DI Water Tank Fill the DI water tank in the main unit with DI water DI water is used to flush sheath fluid ethanol and other cleaning agents from the fluidics system Always check the level in the DI water tank and refill as required before starting shutting down the SH800 and before running ethanol cleaning DI water tank For details about refilling the DI water tank on the fluidics cart see Refilling the DI Water Tank page 149 Tip An alarm is rai
102. Analyzing Data 51 uoneiedo oiseg g13 deyo iii uoleisdo olseg Jsa deuyD 1 000 BSC A 10 104 104 105 106 APC A Compensated 6 Click Edit Statistics in the Statistics group on the Worksheet Tools tab or Plot Tools tab The Statistics Editor dialog appears T Select the statistics to display in the Gates and Statistics table FITC Statistics Editor Equation A AND B Selected Gate A Mean Minimum Maximum SD FSC A Y FSC H 4 BSC A A BSC H A Brilliant Violet 421 A vv Brilliant Violet 421 H FITC A FITC H PE A PE H APC A APC H PerCP Cy5 5 A PerCP Cy5 5 H PE Cy7 A G Select a gate 2 Place check marks in the checkboxes for the Statistics to display 3 Click Apply to update the Gate and Statistics table 4 Click Close to close the dialog The selected statistics are displayed in the Gates and Statistics table Name Events Parent Total Mean Maximum CV D All Events 5 000 0 00 100 00 EID a E A Ba FSC A 8 052 12 27 Pal FSC H 77 516 13 32 Bal BSC A 17 539 fa BSC H 141 886 7 67 a CD56 Brillia 151 91 Bal CD45 FITC 7 981 Bal CD45 FITC 71 213 Mc 1 484 33 15 29 68 e mn es ie B a T T ne B T a T 52 Analyzing Data Sorting Once events have been isolated on plots using gates the same events can be sorted into different collection tubes for further analysis Sorting enables the targeted cells to be recovered from a
103. D 112 Measurement Settings Specifies parameter settings and instrument settings For details about each item see the Measurement Settings dialog page 73 Tube Information Specifies information relating to the sample tubes For details about each item see Tube Information dialog page 135 Compensation Settings Displays compensation settings For details about each item see Compensation Settings dialog page 124 Sort amp Stop Settings Displays the sorting and automatic stop conditions Add Sample Group Displays the Add Sample Group dialog page 112 for adding a sample group to the experiment structure Add Tube Displays the Add Tube dialog page 113 for adding a sample tube to the selected sample group in experiment structure O Delete Selected Item Deletes the selected item from the experiment structure Create New Experiment Creates a new experiment with the specified settings The experiment is added to the Experiment Explorer in the main window The created experiment is automatically made the active experiment Create Experiment Window Add Sample Group dialog The Add Sample Group dialog is displayed by clicking Add Sample Group in the Create Experiment window The Add Sample Group dialog is used to add a sample group to the experiment structure Sample Group Templates list Displays the available sampl
104. Export FCS File Displays the FCS File Export dialog page 117 for exporting the selected sample tube in FCS 3 0 or FCS 3 1 format Delete Deletes the selected sample tube Rename Renames the selected sample tube Show Plate Sorting Monitor Displays the Plate Sorting Monitor dialog page 60 for monitoring the status when sorting onto multi well plates Show Results Displays the Tube Results dialog page 115 for displaying the recording results and sorting results for the selected sample tube Tube worksheet settings The following menu commands are displayed when right clicking a Worksheet Settings icon for a sample tube in the Experiment Explorer Copy Paste Copies the worksheet sheet settings for the selected sample tube and pastes them into the worksheet settings for another sample tube Save as Favorite Saves the current worksheet settings as the favorite default worksheet settings Restore Favorite Restores the saved favorite worksheet settings as the current worksheet settings Apply to All Tubes In Same Sample Group Applies the worksheet settings for the selected sample tube to all sample tubes within the sample group Context Menu Show Settings Displays the Worksheet Settings dialog page 135 for displaying saving and loading worksheet settings Data sources The following menu commands are displayed when right clicking a data source icon in the Experiment Explorer
105. FS format is valid Use only NTFS formatted drives Please use NTFS formatted drive You do not have write permission for the selected folder You do not have write access privileges for the selected Please add write permission to the folder or select another folder folder with write permission Change the folder access privileges or specify a folder with write access privileges Available disk free space is low Insufficient remaining space on the disk Delete unnecessary files to increase the available free capacity This chip has been used over the duration limit 12 hours Replace the sorting chip with the 405 nm laser Please exchange for a new chip This chip has been used over the duration limit 12 hours with the 405 nm laser You cannot use this chip any longer with the 405 nm laser Please exchange for a new chip System resources are scarce and may cause the application Close any unnecessary worksheets performance to be worse Would you like to close unnecessary worksheets A number of plots are opened and may cause the application performance to be worse Please close unnecessary plots or worksheets System resources are scarce and may cause the application Stop data acquisition and measurement and restart the to shutdown Would you like to restart this application SH800 Software Error Messages Ssnosuel aosi g xlpueddy 223 GUI Error Message Recommended Solution System resou
106. In addition the fluidics system should also be cleaned as required after running samples that contain infectious substances Precautions when cleaning with ethanol e Ensure the laboratory is well ventilated during cleaning with ethanol Inhaling ethanol vapor can cause irritation of the eyes skin and respiratory tract loss of coordination drowsiness and in sufficient concentration unconsciousness e Never place a naked flame embers or other material that can emit sparks near the main unit or the fluidics cart Ethanol vapor is highly flammable at normal room temperature e Never pour ethanol into a tank other than the ethanol tank and conversely never pour other liquids into the ethanol tank e Make sure that the cap on the ethanol tank is correctly and securely fastened at all times e Make sure that the ethanol line is securely connected to the connectors on the rear panel of the main unit rear panel of the fluidics cart and the ethanol tank itself e Make sure that the lid on the waste tank is correctly and securely fastened at all times e Make sure that the waste fluid line is securely connected to the connectors on the rear panel of the main unit rear panel of the fluidics cart and the waste tank itself The SH800 is compatible with standard disinfecting agents ethanol 70 used for flow cytometers 1 In SH800 Software click Ethanol Cleaning on the Cytometer tab of the ribbon e The ethanol cleaning sequ
107. Opens the worksheet tab for the tube selected in the Experiment Explorer Apply Compensation Applies fluorescence compensation to the plots on the worksheet for the tube selected in the Experiment Explorer Settings and Information group Contains tool buttons for copying pasting and displaying settings and information 15 A yo sa Copy Faste lt Applyt Shay Snow Show Tubes nronnatic Seitings Results Copy Paste Copies Pastes the item selected in the Experiment Explorer Apply to All Tubes Applies the worksheet settings for the tube selected in the Experiment Explorer to all sample tubes within the sample group Click Worksheet Settings for a tube in the Experiment Explorer and then click Apply To All Tubes to apply the same worksheet settings to the other tubes in the sample group for easy comparison during analysis Show Information Displays the information dialog for the item ia Experiment Information H Sample Group Information etc selected in the Experiment Explorer You can also display the settings dialog by double clicking Experiment Information and other items in the Experiment Explorer Show Settings Displays the configuration dialog for the settings item Measurement Settings Compensation Settings etc selected in the Experiment Explorer You can also display the settings dialog by double clicking Experiment Information and other items in the Experime
108. PE 0 00 0 00 100 00 000 000 0 00 E 8 APC ooo 0 00 0 00 100 00 000 0 00 o7 wis Z PerCP Cy55 1170 06 224 41 33314 5 6625 25 100 00 2650 50 0 00 0 00 0 00 0 00 0 00 100 00 Target Compensation Panel Displays the compensation panel for which to calculate fluorescence compensation Use Negative Value for Compensation Enables calculation of the spillover matrix using a negative control If an appropriate negative control 1s not available deselect the checkbox to calculate the spillover matrix without using a negative control The checkbox default setting is on enabled Spillover Matrix Displays the spillover matrix Spillover matrix values display the fluorescence intensity due to each fluorochrome as a percentage of the total intensity in each channel Calculate Calculates the spillover matrix coefficients Close Closes the dialog Worksheet Tools Tab ribbon The Worksheet Tools tab of the ribbon has the following buttons Plot group Contains tool buttons for adding deleting and duplicating plots T ee we o amp New New Dot New Remove Duplicate Density Plot Histogram Plot Plot For details see Adding a Plot page 83 New Density Adds a new density plot to the worksheet New Dot Plot Adds a new dot plot to the worksheet New Histogram Adds a new histogram plot to the worksheet Remove Plot Removes the selected plot from the worksheet Duplicates Plot Du
109. PEEK sample line soueuajUuley g sajdeyy 144 Parts cleaning Maintenance item Cleaning the e When shutting sample loader down Cleaning the e When shutting sorting area and down transparent protective covers Cleaning the e When shutting droplet camera down windows Cleaning the laser When shutting windows down Cleaning the e When shutting collection tube down holders multi well As required plate holder Cleaning the e When shutting splash guard down e As required Monthly Maintenance Maintenance item Changing Tank Changing the waste tank air filter e Monthly Cleaning the DI water tank and DI water line Monthly As required Running ethanol cleaning Monthly As required Maintenance Schedule General Cleaning page 153 Air Filters page 165 Cleaning the DI Water Tank and DI Water Line page 166 Running ethanol cleaning page 158 PEEK sample line or the sample line Autoclaving the probe adapter and sample probe Changing the sheath filter Changing the DI water filter Changing the sample loader O ring Changing the sample probe Changing the probe adapter Changing the sintered sheath line filter Changing the sheath tank air filter Changing the DI water tank and ethanol tank air filter Changing the waste catcher O ring As required Every 3 months As required Every 3 to 12 months As requi
110. Result Selects print of the sorting results Preview Displays a print preview image on the right side of the screen For details about other items see Print Window page 108 Plot Tools Tab ribbon The Plot Tools tab of the ribbon has the following buttons Refer to the Worksheet Tools tab for a description of the Plot group buttons page 125 Statistics group buttons page 126 Tools group buttons page 127 and Compensation group buttons page 127 Plot Type group Contains tool buttons for changing the type of the selected plot a Density lez Dot Plot m Histogram im Ti an i Scale Type group Changes the type of scale displayed on axes on plots X Axis Lin Ay fp 5 lear uy T YAss Linear Swap ae X Axis Y Axis Changes the type of scale displayed on the X axis and Y axis respectively of the selected plot For details about axis scale types see Changing Axis Scales page 85 Swap Swaps the position of the X axis and Y axis scales la Displays the Property Window dialog page 130 for specifying axis types and scale ranges Axes group Contains tool buttons for adjusting axis scales t TA e i iq 24 Auto Adjust Auto Adjust Auto Adjust Zoom Default My W Axis Y Axis Adjust i Auto Adjust XY Automatically adjusts the ranges of the X axis and Y axis of the selected plot to their minimum and maxi
111. SH800 icon in the task bar to minimize the main window and then click it again to restore the window Troubleshooting snosuel eosiy g xipueddy 217 snosuel aosi g xlpueddy 218 Error Messages This section lists the error messages that may be displayed in SH800 Software When an error occurs check these items for the actions to take to resolve each error If the problem still persists after carrying out the recommended actions make a note of the error message and then contact your Sony distributor GUI Error Message Recommended Solution Unload Chip Error Discard and load a new chip If the error reoccurs please contact your service representative Load Chip Error Error on loading mechanism Please reinsert the chip If the error reoccurs please contact your service representative Host Communication Error 1 Shutdown the cytometer 2 Make sure the cable is connected correctly and firmly and restart the cytometer and software lf the error reoccurs please contact your service representative Unload Sample tube Error If the error reoccurs please contact your service representative Unload Sample tube Error Failed to reduce the sample pressure If the error reoccurs please contact your service representative Unload Sample tube Error Failed to down the sample loader If the error reoccurs please contact your service representative Unload Sample tube Error Failed to move the
112. SONY Cell Sorter Operator s Guide Software Version 1 7 LE SH800 Series C 181 100 11 1 Chapter 1 Overview Chapter 2 Preparation Chapter 3 Basic Operation Chapter 4 Configuring Experiments Chapter 5 Analysis Chapter 6 Sorting Chapter 7 Window Description Chapter 8 Maintenance Appendix A Operating Principles Appendix B Miscellaneous Index 2 Table of Contents Software Revision History ccssscceseeceeeeeeeeseeeeeeeeeeeneeees 8 Using the PDF Manual ccccccessceseseeeeeeeeseeeeeeeeeseeseaseneaees 9 Model Identification sissccacsuanninwonvcusvsnaonincsiaweenaasveareceuucraiuieons 10 Components and Documentation ccsseceseeesseeeneeenees 10 Chapter 1 Main Features cpccasccsacasheaaceuvactinaticussvacauevacsbeusborianspiaanonenaubtens 12 Cell Sorter Block Diagram c cccsesseeeceeseeeeeeneeeseeeneees 14 System Configuration cscccssssscssssseecsnsseeceneseseeesseseens 15 Name and Function of Parts ccsscesseeseeeseeeseeeeeeneeees 16 FOCE ooren E E 16 Internal View PRONT seee a TER 17 MISC VIEW dE esre e s 21 Rear pane biracisorn ana Sobiasvapediasenawtias 22 PUC C aesan a a 23 SORAS GS 6h a arene pe ome ee Cen rere pen rs oe en ce Pyrenees Te 29 Man NA OW aaan T 25 SH800 Software Operating Environment 000 27 EGD MONITO sroronionnaa a ai 28 Chapter 2 Preparation Table of Contents
113. Sheath Line Filter soueuslUle g Ja deyuy I 179 soueuajUle g sajdeyyD 180 4 Inthe same way remove the white connector on the sheath tank side and discard the filter Changing the Sintered Sheath Line Filter Discard the sintered sheath line filter in accordance with local and federal ordinances and regulations Insert a new sintered sheath line filter in the sheath fluid line pushing the line all the way into the filter then connect the white connector to the sheath tank Releasing Air in the Sheath Filter and Sorting Chip During operation air may become trapped in the sheath filter interrupting the smooth flow of sheath fluid to the sorting chip Air may also become trapped in the sorting chip itself preventing the formation of regular shaped droplets To release air trapped in the sheath filter using the de bubble function 1 Click Sheath Filter in the De bubble group on the Cytometer tab of the ribbon The Sheath Filter De bubble dialog appears 2 Click Start and then click Close when finished The sheath filter de bubble process takes a short time to finish If changing the sheath filter run the sheath filter de bubble process at least three times to make sure that all air and small particles have been removed from the filter and the sheath line To release air trapped in the sorting chip using the de bubble function If the formation of droplets is irregular even t
114. Sort Settings Specifies the default sorting configuration Clicking this button displays the Sort Settings dialog For details about the Sort Settings dialog see Sorting page 53 and Adjusting the Sort Position SHSOOZP only page 99 Sort Statistics pane Displays the statistics about events during sorting Total Elapsed Time Displays the cumulative elapsed time since the start of sorting of the whole plate Total Progress Displays the ratio of sorted wells to the total number of wells Sort ID Displays the ID of the well currently being sorted Well Number Displays the number of the well currently being sorted Sort Mode Displays the sorting mode of the well currently being sorted Cell Size Displays the current cell size setting Sort Gate Displays the sort gate of the well currently being sorted Stop Count Displays the number of events for the automatic stop condition of the well currently being sorted Plate image Displays the layout of the wells Double clicking the plate image or right clicking and selecting Show Sorting Report from the context menu displays the plate image in a separate window for monitoring progress Elapsed Time Displays the cumulative elapsed time since the start of sorting Remaining Time Displays the estimated remaining sorting time Sort Count Displays the number of events sorted Sort Rate Displays the rate at which events a
115. The cytometer has been disconnected Check the connections to the main unit Acquiring and sorting has stopped Communication to the cytometer has been lost Check the connections to the main unit and relaunch SH800 Exit and relaunch the software Software The initial setup did not complete successfully Exit SH800 Software and try launching again Exit and relaunch the software Acquiring and sorting has stopped because available free Insufficient remaining space on the hard disk Delete size of Hard Disk Drive is not enough unnecessary files to increase the available free capacity The application is already running This instance will now Shut down the SH800 Software instance that is already exit running Failed to save settings Try saving the settings again Parameter count in a selected file is different from current Select a file with the same number of parameters as the settings current settings A selected file is wrong format Select a file in the correct format Unable to maintain stable droplet breakoff An error occurred when the control breakoff function was turned on If a droplet formation problem is detected during measurement acquisition stops Try running Sort Calibration If the problem persists contact your Sony distributor for service The selected drive is not external storage device Specify an external storage device Please select the external storage device The format of this drive is invalid Only NT
116. Vv 1 Gain Settings v Negative control Brilliant Violet 421 Data acquisition starts for the tube and data points are added to the plots on the worksheet in real time A Click Detector amp Threshold Settings Pause Restart Stop Record Sampie Stop Condition Detector amp Threshold Settings Sample Pressure 4 Y The Detector amp Threshold Settings dialog appears Performing Fluorescence Compensation 61 uoneiedo oiseg g13 deyo Kii uoleisdo olseg sa deyuyD 5 Adjust the sensor gain of the FSC and BSC detectors to place the population on scale Detector amp Threshold Settings Threshold Channel Value Sensor Gain ee 30 0 to Hja FLL FL2 FL3 FLA FLS PE APC PerCP Cy5 5 PE Cy AO ry d iT fm AO Do m Meo Ay e 40 0 ae 40 0 are An ne 40 055 AN roe 40 05 F fi i Dr 40 0 a leet Fed Haa ees Hja You adjust the gain while viewing the FSC vs BSC density plot You can also adjust the axis display scales to display a magnified portion of the plot 6 Adjust the trigger threshold level to remove background noise without affecting the targeted population Detector amp Threshold Settings Threshold Sensor Gain FSC BSC FLI FL2 FL3 FLA FLS PE APC PerCP Cy5 5 PE Cy m gm m r wat 20 0 ep m ee al Fall oC 40 055 40 0 m mnr f Cy 40 0 eer 400
117. aS SS E Start 8 Click Sort Start in the Sort Control pane to begin sorting v Sort Control Ka 8s VAN Sort amp Record Start Load Collection Sorting Method amp Well Strips v To Sort S Sort Settings Stop Val Tip Starting sorting automatically starts sorting and recording Basic sorting statistics are displayed together with progress bars if an automatic stop condition is specified Total Elapsed Time 00 00 00 Sort ID Well Number A Elapsed Time 00 00 00 c Remaining Time 00 00 00 Sort Mode Sort Count 0 p _ _ _ _ _ _ _ gt Cell Size Sort Rate 0 eps Sort Gate nEs ad Sort Efficiency 0 Stop C a A 0 eps Total Progress 0 2 Double clicking the image displays the side streams in a Separate window for monitoring sorting Droplet Viewer Q x075 e o 0 Double clicking the plate image or right clicking and selecting Sorting Monitor from the context menu displays the Plate Sorting Monitor window for monitoring progress You can also display the window by right clicking a tube in the tube list and selecting Show Sorting Report from the context menu Plate Sorting Monitor Sorting Method 8 Well Strips Sort Statistics Total Elapsed Time 00 00 58 Total Progress 16 16 Plate Number 1 1 A r Q bax a i B Oo D O Sl if O k O m m c 10 10
118. ag it to the desired location Tip The Name column cannot be moved To export statistics as a CSV file Click Export Statistics to CSV File in the Statistics group on the Worksheet Tools tab of the ribbon orksheet Tools Plot Tools Gate Tools 1 Data Source 1 All Events B ET gt VA te GQ HA FA T Remove Edit Show Edit istics Show Snap to Grid Gate Gate Tabie Statistics Grid The Exporting statistics table dialog appears 2 Click Browse to specify the folder to save the CSV file then click Export Tube 2 Export statistics table Output Path The CSV file is exported 3 Click Close to close the dialog Sorting Chapter This chapter describes the various sorting modes and detailed sorting criteria The SH800 can sort selected populations of cells at high speed It sorts droplets containing targeted events selected using gates on plots in the worksheet into left and right output streams two way sorting Sorted droplets can be collected in 5 ml round tubes 15 ml conical tubes and on multi well plates In addition gates can be combined using Boolean logic to uniquely identify a population for sorting using multiple attributes providing powerful sorting capabilities Sorting Mode Overview In an ideal sort cells travel past the optical detection point in single file and are sorted one cell at a time in separate droplets In practice however cells
119. alibration There may be air bubbles trapped in the sheath filter Remove the air bubbles or automatic calibration stops and does from the sheath filter See Releasing Air in the Sheath Filter and Sorting Chip not proceed page 181 If an error occurs while adjusting the side streams the sensor windows may be dirty preventing normal side stream detection Clean the windows using a soft lint free cloth lightly moistened with water Clean the black sensor windows on the left right and back The temperature within the unit may have changed With the main unit up and running leave standing until the room temperature stabilizes then run automatic calibration again If the problem persists there may be salt deposits in the flow channels Log off and run the unit in maintenance mode Running Maintenance Mode page 158 e f the problem persists change the sorting chip e If the problem persists contact your Sony distributor for service Side streams are not visible during The transparent protective covers in the sorting area may be charged with automatic calibration static electricity Clean the covers using a soft lint free cloth moistened with water For details see General Cleaning page 153 The output pressure of the air Connect the power cord for the air compressor directly to a power outlet Do not compressor drops connect using an extension cord The waste tank is bulging The waste tank air filter may be blocke
120. and page 137 e Improved accuracy in sorting position adjustment for multi well plate sorting four corners of plates page 99 Additional test modes for multi well plate sorting page 100 Added option to skip rinsing of the outside of the sample probe during cleaning using DI water page 106 e Added multi well pattern selection buttons for when configuring 8 well strip and multi well plate sorting page 56 and page 59 Software Revision History Functions Added in Version 1 6 The following functions have been added in SH800 Software version 1 6 Support for 130 um sorting chips on all models Added support for 130 um sorting chip even for 2 way sorting on 96 well plate models For details see Sorting page 53 Support for AMS Aerosol Management System evacuators SH800Z and SH800ZP A S A F E System evacuator from Edge Systems LLC can now be connected using a specialized adapter For details see Connecting an AMS Evacuator SHSOOZ and SHS00ZP page 213 Large Cell can be selected in all sorting methods Large Cell can now be selected in 8 well strip slide glass and 96 well plate sorting methods in addition to the 2 way sorting method Functions Added in Version 1 5 1 The following functions have been added in SH800 Software version 1 5 1 Supported by the SH800 The following functions were added e Index sorting e Specifying a gate as calculation target for fluoresce
121. annel FSC v m 488nm On Turn Off Value 5 00 ean i 561nm On Turn Off Sensor Gain i FSC 4 asc 30 0 F 638nm Off Turn On Low es s La Fi 4o0o H rz 40 0 H Temperature Controi Ce a Sample 5 v C FL3 40 0 al FLA 40 0 gt Collection 5 g F5 40 0 H Fe 40 0 gt Fa 7 Probe Wash Unit Celsius v Skip rinse outside of sample probe with DI water Advanced Settings A Review the instructions in the Operator s Guide before changing options in Advanced Settings loa lo ft The Advanced Settings dialog appears On the Pressure Options tab click Standby to stop the sheath fluid flow Advanced Settings Droplet Control Sort Options Stream Optionsf Pressure Options Drop Drive 0 000 Drop Clock 33500 H Hz Sort Delay 30 H Sort Phase 200 H deg Flow Control Sample Pressure a5 S a 2009 psi Sheath Pressure 138 0 a 2001 psi Correction 9 or or o 0 Calibration Display Mode ee 0 0 Save Droplet Info Deflection Control Stream Deflection Collection Stage Load Collection Waste Catch Main Side Mid Main Sort Test Sorting Stream Right Start Sorting Stream Left Waste Stream 2048 o H4 2048 Charge Peak 0k Perform steps 3 to 8 in Refilling the Sheath Tank page 146 to remove the lid of the sheath tank 7 Dispose of the remaining
122. anol cleaning or bleach cleaning without needing to log in to SH800 Software For details see Running Maintenance Mode page 158 Using the PDF Manual The Operator s Guide can be read on a computer with Adobe Reader installed You can download Adobe Reader free from the Adobe website 1 Open the Operator s Guide folder in the installation disc 2 Select and click on the Operator s Guide that you want to read If you have lost or damaged the installation disc you can purchase a new one from your Sony distributor Using the PDF Manual 9 Model Identification The following model names are used to refer to the various unit models in this document SH800 LE SH800AC BC Supports collection CC DC EC F tube 2 way 8 well SH800Z LE SH800ZA ZB or slide ZC ZD ZE ZF ZG 9 ZH SH800ZP LE SH800ZAP ZBP ZCP ZDP ZEP ZFP ZGP ZHP Supports collection tube 2 way and multi well plate 3 1 SH800 Software 1 7 supports both PEEK sample line compatible models and non PEEK sample line compatible models See Sample Line Support below 2 SH800 models upgraded to support 96 well plate sorting are equivalent to SH800ZP models 3 In this document 6 12 24 48 96 and 384 well plates are collectively referred to as multi well plates or 96 well plates Sample Line Support PEEK sample line with probe Sample line sample probe metallic PEEK sample
123. aration for data acquisition e Start Compensation Wizard Launches the Compensation Wizard to perform fluorescence compensation File Window Explorer Expand Public User s data Selects whether to display the Public user data e On e Off Export Experiments Default Export Folder Specifies the destination folder used when exporting experiment data Worksheet tab Defines default settings related to the worksheet User Preference Sort Set default settings for worksheet Reset Cytometer Plots i Set default displayed events during acquisition N isplay Events in Acquiring 5 000 Compensation i Set default events displayed during analysis o Displayed events nn alysis 00 User Defined Color nts during analysis 100 000 FCS File aoo i agg Set default color palette for plots EEE Color Palette Plots Display Events in Acquiring Selects the number of events to display during data acquisition Display events during analysis Selects the number of events to display when analyzing data Color Palette Selects the color palette background color and display of outlying rare events e Black Rare e Black Normal e White Rare e White Normal Gate Label Font Size Selects the font size of text labels shown on gates You can check the size in Preview Gate Line Width Selects the line width used to draw gates You can check the line width in Preview Auto
124. arted End Time Displays the time when recording ended Compensation Settings Previews restores fluorescence compensation settings saved when data recording ends e Preview Displays the fluorescence compensation settings e Restore Restores the fluorescence compensation settings Click Yes in the confirmation dialog to proceed Worksheet Settings Previews restores worksheet settings saved when data recording ends e Preview Displays the worksheet settings e Restore Restores the worksheet settings Click Yes in the confirmation dialog to proceed Instrument Settings Displays the instrument settings Main Window e Sorting Result tab Displays the sorting results Copy to Clipboard Copies the sorting results data to the clipboard as an image for pasting in other software for example to produce reports Save as CSV Saves the sorting results as CSV format file Compensation Settings Previews restores fluorescence compensation settings saved when sorting ends e Preview Displays the fluorescence compensation settings e Restore Restores the fluorescence compensation settings Click Yes in the confirmation dialog to proceed Worksheet Settings Previews restores worksheet settings saved when sorting ends e Preview Displays the worksheet settings e Restore Restores the worksheet settings Click Yes in the confirmation dialog to proceed Sort Settin
125. aseiincetorl n 144 Miscellaneous gsi casctaws acu Ura a a 145 PIUIGICS Caic a 145 Refilling the Sheath Tank cccccsssseesseseeeeesseeeeenseeeeeneees 146 Emptying Changing the Waste Tank sssecsssseseeees 148 Refilling the DI Water Tank ccccsessceseseeeeeseeeeeneeneeneeees 149 Refilling the Ethanol Tank cccccccsseseeseseeeeeseeeeeeeseeaeeaees 151 Cleaning the Sample Fluidics System using Bleach 152 General Cleaning ssecsceccicccowsscccestncedeunseensseteectssriecsns danadana 153 Cleaning the Deflection Plates ccssssseeseeseeeeeeneeeeees 155 Cleaning the Waste Catcher cssscceeseeeceeseseeeeesensees 157 Running Maintenance Mode ccccsseseeneseeeeenseeeeneeneees 158 Changing the Sample Loader O Ring sssssseeeeeseeeees 160 Cleaning the Sheath Tank and Sheath Line 162 Changing Tank Air Filters cccccssssseeesesseeeeenseeeeeneenees 165 Cleaning the DI Water Tank and DI Water Line 166 Changing the PEEK Sample Line PEEK sample line compatible models scccescceeeeeseeeseeeeseenseeeeeees 169 Changing the Sample Line and Probe Non PEEK sample line compatible models 00s 00000 172 Changing the Probe Adapter PEEK sample line compatible models seceeseceeeeeseeeseeeeseeeseeeeeees 175 Changing the Sheath Filter cccssssccssseecseeseseeseesensee
126. aste tank 23 Fluidics cart air line connector 23 24 Fluidics cart connectors 23 Fluidics maintenance door 21 Fluorescence compensation see compensation Fluorescent light FL 196 Fluorochrome detection matrix 204 Forward scatter FSC 196 H Heat exhaust vent 22 Hydrodynamic focusing 193 Initial Instrument Setup screen 43 Installation disc 9 L Laser configuration 44 light collection 198 source 197 wavelength 201 LCD monitor 17 28 Login 42 Longpass optical filter LPF 198 M Main power switch 22 Maintenance 143 Maintenance mode 158 O Operation principles 191 Operation flow 40 Optical filter cleaning 186 pattern 202 Optional accessories 225 P Parts replacement 225 Password changing 39 PC connection cable connector 22 Pinch valve 17 Power turning on 41 POWER STANDBY button 16 Pulse parameter description 199 Q QR code 43 R Replacement parts 225 S Sample fluid flow 192 Sample line 17 changing 172 Sample loader Door 16 Door button 16 O ring 160 Sample probe changing 172 Scatter light 196 Sheath filter 21 autoclaving 183 de bubble 181 replacing 176 Sheath fluid flow 192 Sheath line cleaning 162 Sheath line filter 179 Sheath tank 24 cleaning 162 refilling 146 Side stream calibration 46 Side stream monitor Image 46 Slide glass collection 19 33 Software license 225 Sort delay calibration 46 Sort parameters 103 Manual adjustment 103 Sorting 53 Adjusting 99 mod
127. asteA maintenance Click Start to initiate WasteA maintenance WasteA maintenance will take about 10 minutes For details about cleaning using Sodium Hypochlorite solution see Cleaning the Sample Fluidics System using Bleach page 152 When WasteA maintenance finishes the login screen reappears Changing the Sample Line Click Maintenance on the bottom right of the SH800 Software login screen Cell Sorter SH800 The Maintenance wizard appears Running Maintenance Mode soueuslUle 813 deyo I 199 soueuajUleW g Jajdeyuy 160 2 Select Sample line exchange then click Start Maintenance v Internal sheath line and DI water line are cleaned with ethanol WasteA maintenance It is recommended that if you have not used for a long time to perform WasteA maintenance Sample line exchange OJ Exchange sample line and probe for new one The Sample Line Exchange wizard appears 3 Click Start to begin changing the sample line Follow the on screen instructions Sample Line Exchange Click Start to initiate Sample Line Exchange procedure Note The installed chip will be unloaded after Sample Line Exchange procedure is initiated For details about changing the sample line see Changing the PEEK Sample Line PEEK sample line compatible models page 169 or Changing the Sample Line and Probe Non PEEK sample line compatible models page 172
128. ata recording starts If an automatic stop condition was specified data acquisition and recording stops automatically when the stop condition has been satisfied Q fan automatic stop condition was not specified None click Stop to stop data acquisition and recording The sample tube unloads automatically Acquisition Tab Main Functions During data acquisition the data is displayed on plots and in the Gates and Statistics table on the worksheet You use the buttons on the Acquisition tab as required to control data acquisition while monitoring the data acquisition status on the worksheet You can change the type of plots edit gates on plots and customize the content displayed in the Gates and Statistics Controls data acquisition and recording Plots Displays acquired data in graphical form Avent 8 8 ee ae Ha B a o se E B ol Sr R mi BB Gates and Statistics i Droplet Hardware Status Displays the experiment structure hierarchically Gates and Statistics table Displays statistics calculated automatically from acquired data for each gate Controlling Data Acquisition and Recording After data acquisition starts the elapsed time since the start of data acquisition the total number of events acquired and the event rate measured in events second eps are displayed on the Acquisition tab You control acquisition as required while monitoring the acquis
129. ate is added to the plot together with a single statistic for the gate Example Polygon gate Statistic Example Linear gate Tube 2 Index 96 Well Data Source 1 Tube 3 finde 3 Edit the name of the gate the displayed statistic color and other preferences as required Select the gate click Edit Gate in the Gate group on the Plot Tools tab of the ribbon to display the Gate Editor dialog and then select the Details tab FITC Gate Editor For details about each item see Gate Editor dialog page 127 Copying a Physical Gate You can copy a gate drawn on a plot to another plot 1 Right click a gate to copy then select Copy Region from the context menu Tube 1 Negative control Brillian All Events Ci Create Density Plot P Create Dot Plot ai Create Histogram Plot Convert to Dy Copy Region Ctri C we amp Visible Gate G Remove Delete HE Send To Back Properties 2 Paste the gate on the target plot Tip You can also right click a gate in the Gates and Statistics table to copy a gate within the gate hierarchy using commands in the context menu Adding a Boolean gate to the Gate Hierarchy Selecta plot to display the Plot Tools tab 2 Click Edit Gate in the Gate group on the Plot Tools tab of the ribbon A C Cov Edit Export to Statistics CSV File Croat HESUS A E e amp Polygon Quadrant Linear Remove Gate b
130. ath filter Tip The sheath filter de bubble function should be run at least three times after changing the sheath filter Auto Calibration group Contains tool buttons for aligning the chip and calibrating sorting aT Sort thio Caibration Alignment Sort Calibration Displays the Auto Calibration wizard for calibrating sorting For details see Automatic Calibration page 46 Chip Alignment Displays the Chip Alignment screen of the Auto Calibration wizard for aligning the sorting chip Follow the on screen instructions to align the sorting chip During automatic alignment the progress bar and status are displayed allowing you to check the progress and current state Temperature Control group Contains tool buttons for controlling the temperature of the collection stage and sample loader to maintain sample fluid temperature The following temperature control options are available e Collection stage 5 C 41 F only e Sample loader 5 C 41 F or 37 C 98 6 F boy y Collection Sample 5c gac Collection Turns the collection temperature control on off and displays the current temperature setting Sample Turns the sample loader temperature control on off and displays the current temperature setting Clicking the a dialog launcher displays the Temperature Control Settings dialog page 120 for specifying the temperature settings Agitate group Contains a tool but
131. ault behavior by user preference Start acquiring first tube Displays the main window and assigns the first tube in preparation for data acquisition Start compensation wizard Displays the Compensation Wizard to record control tubes for fluorescence compensation For details about the Compensation Wizard see Performing Fluorescence Compensation page 61 The experiment is created and the main window appears The created experiment is automatically made the active experiment and the first tube is automatically assigned as the active tube Comgersaton compensation To create an experiment using an existing experiment 1 In step 2 4 of the procedure above select an experiment you want to use from Recent Experiments instead of a template Recent Experiments displays the most recently used experiments in reverse chronological order Wg OO 2 Check the experiment details and enter a name for the experiment in Name under Experiment Information 48 Creating an Experiment 3 Click Create New Experiment The experiment is created and the main window appears To save an experiment as a template You can save an experiment as a shared template Public Templates or a private template My Templates For details see Saving an Experiment as a Template page 78 Acquiring Data After an experiment is created you acquire and record data for
132. b on the left of the main screen Click Export to FCS file in the Export group on the Experiment tab of the ribbon FCS Export to Fics file Export The FCS File Export dialog appears Place check marks in Select target tubes for tubes whose data is to be exported specify the FCS format and export destination folder then click Export You can also select the required data source if there is more than one data source for a single tube FCS File Export Select target tubes Experiment 2014 01 16 16 15 11 4 Sample Group 1 av 2 Tube 7 a 8 Index 96 Well Data Source Y 6 Index 96 Well Data Source sav 2 Tube 2 SEXP A 6 Index 96 Well Data Source A Index 96 Well Data Source The tube data is exported The name of the exported file is the same as the name of the tube with an fcs filename extension e Convert BSC to SSC Selects whether to change the sensor name for data recorded by the BSC back scatter detector to SSC side scatter when exporting FCS files T Searching Experiments Saving Tube Data as FCS Files sluewuedxy Buunbyuoy p Jaldeyy HNN s u wn dx Buunbyuog p Je deyD 78 e The required FCS text segment keywords are exported automatically The following optional FCS text segment keywords can also be specified INST Institution at which data was acquired EXP Name of investigator initiating the experiment Name of flow cytomete
133. bar Displays the export progress Main Window Export Starts the export Close Closes the dialog Exporting Data dialog The Exporting Data dialog is displayed by clicking Export Data to CSV File on the Worksheet tab of the ribbon The Exporting Data dialog is used to export recorded data for sample tubes as a CSV format file Tube 1 Exporting Data Output Path Output Path Specifies the export destination folder Click Browse to specify for a destination folder Progress bar Displays the export progress Export Starts the export Close Closes the dialog Custom Print dialog The Custom Print dialog is displayed by clicking Custom Print in the Print group on the Worksheet Tools tab of the ribbon The Custom Print dialog is used to select items for printing Print Print Settings NorthAmericatetter Custom Settings Selects the sample tubes for printing Select the checkbox for a tube to print the worksheet for the tube Print Experiment Information Selects whether to print the experiment information settings Print Tube Information Selects whether to print the sample tube information settings Print Sort Settings Selects print of the sort settings Print Worksheets Selects printing of the worksheet settings Print Statistics and Record Result Selects printing of the Gates and Statistics table and recorded results Print Sort
134. ble Statistic to CSV N el D Display Events Sets the number of events displayed on plots Gate Tools for editing gates Statistics Shows hides the Gates and Statistics table customizes the content displayed in the table and exports the table as a CSV file Worksheet View Changes the view and scale of plots on the worksheet For details see Worksheet Tools Tab ribbon page 125 Analyzing Data After data acquisition finishes you analyze the acquired data events The most recent data is displayed on the worksheet immediately after acquisition finishes To analyze data for a past experiment search for and select the target experiment on the Experiments tab of the Experiment Acquisition control pane Plots Displays acquired data in graphical form Plots can be added edited Gates Groups events with common characteristics isolating them from all other events Experiments tab Displays experiments that can be selected for analysis Gates and Statistics table Displays statistics calculated automatically from acquired data for each gate You can customized the statistics displayed in the table This section describes how to add plots to the worksheet how to add gates to a plot and how to display statistics for the gates For details see Analysis page 83 1 Select the All Events plot on the wo
135. bon The bleach cleaning sequence must be followed to completion once it is started 2 Load a 15 ml conical sample tube containing 10 ml of 1 to 3 Sodium Hypochlorite bleach solution in the sample loader Tip When performing bleach cleaning during measurement perform the cleaning sequence once using a 15 ml conical sample tube When performing bleach cleaning before shutdown at the end of a day perform the cleaning sequence three times using a 15 ml conical sample tube or once using a 30 ml cleaning tube 3 Click Start to begin the cleaning process The solution passes through the sample probe sample line and sorting chip A progress bar is displayed during cleaning Abort the process if necessary before the level of bleach in the sample tube falls below the tip of the sample probe 4 When finished remove the tube 5 Click DI Rinse on the Cytometer tab of the ribbon 6 Load a 15 ml conical sample tube containing 12 ml of DI water in the sample loader The level of DI water must exceed the level of bleach added in step 2 Click Start to begin the rinse process Stop the DI rinse process if necessary before the level in the sample tube falls below the tip of the sample probe 8 When finished remove the tube 9 Click Probe Wash on the Cytometer tab of the ribbon to flush the sample line and probe Always use 15 ml conical tubes with the specified fluid volume when using the Bleach Cleani
136. button on the front panel of the main unit 20 Launch SH800 Software On the Auto Calibration screen click Skip Auto Calibration to bypass automatic calibration 21 Click Sheath Filter in the De bubble group on the Cytometer tab of the ribbon A popup window appears 22 Click Start to begin cleaning then click Close when finished Repeat this procedure at least five times to remove all traces of ethanol in the sheath fluid line 23 Disconnect the filter bypass line and reconnect the sheath filter lines 24 Perform steps 3 to 6 in Refilling the Sheath Tank page 146 to remove the sheath tank from the tray 25 Grasp the handles and shake the sheath tank to clean the inside of the tank as described in step 8 26 Remove the lid of the sheath tank and dispose of the DI water Dispose of the DI water in accordance with the laboratory rules and local ordinances and regulations 2T Refill the tank with sheath fluid For details see step 7 and later in Refilling the Sheath Tank page 147 Changing Tank Air Filters The DI water tank in the main unit and the ethanol tank and waste tank in the fluidics cart are each fitted with an air filter The filter allows air to flow into or out of the tank as the fluid level in the tank changes The air filters on the three tanks are connected using the same type of connector This section describes the replacement procedure using the DI water tank
137. click Start i Normal Use 15 ml tube with 12 ml DI water Careful Use 30 ml tube with 30 ml of DI water Start Close Sheath Filter De bubble dialog The Sheath Filter De bubble dialog is displayed by clicking Sheath Filter in the De bubble group on the Cytometer tab of the ribbon The Sheath Filter De bubble dialog is used to remove air bubbles from the sheath filter Click Start to begin the process Sheath Filter De bubble Select Start to de bubble the sheath filter Start Close Tip The sheath filter de bubble function should be run at least three times after changing the sheath filter Temperature Control Settings dialog The Temperature Control Settings dialog is displayed by clicking the w dialog launcher in the Temperature Control group on the Cytometer tab of the ribbon The Temperature Control Settings dialog is used to set the temperature of the sample loader and collection stage area Temperature Control Settings Sample a Y m a a 5 k ar CONECTION L Unit Celsius iv Close Sample Sets the control temperature of the sample loader to 5 C 41 F or 37 C 98 6 F The selected temperature values are displayed on the corresponding buttons in the Temperature Control group on the ribbon Collection Selects the control temperature of the collection stage area Only 5 C 41 F is available Unit Selects the units of temperature Celsius or Fahrenhei
138. close the door V 2 Chip Information 3 Chip Loading The fluidics check and automatic calibration sequence begins Follow the on screen instructions to align the chip and calibrate the instrument e An error message appears if the sorting chip is inserted in the incorrect orientation e Do not insert any objects other than Sony sorting and cleaning chips designed for the SH800 into the chip insertion slot e The packaging for the loaded sorting chip should be stored in a safe location e Replace the sorting chip if it is discolored dirty or stained from previous use e Do not drop the sorting chip on a hard surface or bend the chip intentionally If there is a problem with the sorting chip contact your Sony distributor e Always wear gloves and other protective clothing mask and goggles when changing the sorting chip e If the sorting chip will not load correctly run the instrument in maintenance mode as described in Running Maintenance Mode page 158 to clean the instrument If the problem persists contact your Sony distributor for service e The sorting chip must be changed regularly if subjected to prolonged exposure to irradiation by the lasers to prevent burn in especially by the 405 nm laser You can monitor the length of time the chip has been exposed to the light from each laser in the Chip Information tab of the Cytometer Settings dialog accessed by clicking Settings on the Cytometer tab of t
139. commended for reliable sorting Select the size of the cells to be sorted in Cell Size As a rough guide select Regular Cell for cells up to 15 um in size and Large Cell for larger cells When you select Large Cell a charge setting suited for sorting larger cells is used However this function does not guarantee complete accuracy in sorting large cells Enter the number of events to sort in Stop Count If a value exceeding the values below is entered in Stop Count a message appears warning you of the risk that sample fluid may overflow the well operation does not stop 96 Well Plate 50 000 events Enter the timeout time in Timeout in units of seconds Click Add The settings are added to Sort ID List Repeat steps to to set parameters for all wells You can modify the parameters for a well by clicking the corresponding cells in Sort ID List 4D When finished click Close to close the dialog For details about the Plate Adjustment tab see Adjusting the Sort Position SHSOOZP only page 99 Click Start or Resume in the data acquisition control pane to begin data acquisition Status Ready 00 00 00 Total Event 0 FHlapsed Time Event Rate 0 eps pne eS E Start Click Sort Start in the Sort Control pane to begin sorting v Sort Control Sort Sta Total E re eS vy Total F Sort amp Record Start Load Collection Sorting Method
140. d Replace the waste tank air filter See Changing Tank Air Filters page 165 Water splashes occur when cleaning the De bubble the DI water filter sample probe For details see Changing the DI Water Filter page 177 The instrument will not operate after Adding a printer in Control Panel while the SH800 main unit and host computer adding a printer in Control Panel on the are connected may cause the SH800 main unit to stop functioning Press and host computer hold the POWER STANDBY button on the front panel to turn off the power then turn the power on again and restart SH800 Software To add a printer first turn off the SH800 power supply or disconnect the network cable before adding the printer The main window in SH800 Software If the main window display does not update click the SH800 icon in the task bar does not update e g the event rate does to minimize the main window Then click the SH800 icon again to restore the not update window If pull down menus still do not open in the main window open the Detector amp Threshold Settings dialog page 79 and operate a Threshold pull down menu for example Button labels displayed for dialogs on the If the dialog display is corrupted press the ESC key to close the dialog clicking main window are transparent the X icon in the top right corner of the dialog has no affect Then reopen the dialog from the main window If the problem persists click the
141. d Conflicting cell in the adjacent droplet DEDEDE Sorting Mode 97 uos 9g Jajdeuy Semi Yield Mode Yield Mode In Semi Yield mode a droplet is sorted if it contains one In Yield mode a droplet is sorted together with the or more targeted events even if the droplet contains adjacent droplet nearest to the target event if it contains conflicting events In addition if a targeted cell is near one or more targeted events even if the droplets contain the edge of the droplet the adjacent droplet is also sorted conflicting events even if it too contains conflicting events Two droplets are sorted for each event matching the One or two droplets are sorted for each event matching sorting criteria the sorting criteria 80 Sorted Target cell in edge of droplet so 5 Nx adjacent droplet also sorted z lt gt lt gt Xy X Sorted Target cell in edge of droplet so adjacent droplet also sorted even though it contains conflicting cell 4 ar 2 JODL t aD Sorted Target cell in droplet even though 7 J it also contains conflicting cell gt ap ar Q8 Sorting Mode Ultra Yield Mode In Ultra Yield mode a droplet is sorted together both adjacent droplets if it contains one or more targeted events even if the droplets contain conflicting events Three droplets are sorted for each matching sorting criteria CO Ca BDO O OT TD
142. d to improve user work flow e Flow channels embedded within the chip for precision sheath fluid and sample fluid flow control e High uniformity droplet formation e Automated chip loading and unloading system e Easy optimization of system sorting and analysis conditions when changing sorting chip Main Features Chapter The SH800 interrogates cells in samples using up to four lasers 405 nm 488 nm 561 nm 638 nm depending on the model It measures forward scatter back scatter and six channels of fluorescent light The wide range of excitation wavelengths supports the use of a wide variety of common fluorochromes for use in cell analysis The SH800 can measure intrinsic properties such as size and internal complexity using forward scatter and back scatter analysis It can simultaneously measure a wide range of extrinsic properties using fluorescent light emission analysis High speed electronics allow the operator to specify multiple gates that isolate cells with defined characteristics for further processing Gates can be applied to provide multi parameter criteria for real time identification of selected cell populations at speeds of 100 000 events per second eps for analysis and up to 10 000 eps for sorting The SH800 supports high speed two way sorting to isolate populations for further study or testing An array of automated mechanisms is incorporated to enable easy use by non specialist operators These
143. d within the same droplet or in adjacent droplets The sorting mode selection in SH800 Software determines whether to sort or abort droplets whenever these coincidences occur See Sorting Mode page 93 The time taken for droplets to traverse the distance between laser interrogation at the optical detection point and the breakoff point is called the sort delay The sort delay controls the timing of the electrostatic charging of the target droplet for stable sorting The droplet camera monitors the droplet stream before and after the breakoff point The stroboscopic output image of the droplet camera is displayed in the bottom of the main window in SH800 Software The output image is also used by control circuits to automatically adjust the Breakoff point High voltage deflection plates on either side of the droplet stream deflect the charged droplets using electrostatic attraction and repulsion to the left and right as the droplets fall between the deflection plates sorting the droplets into two side streams two way sorting The polarity of the charge on each droplet determines the direction of the stream left or right Uncharged droplets main stream are not deflected and fall into the waste catcher The side streams are directed toward the collection tubes on the collection stage Left side stream The deflection plates are charged at extremely high voltages Contact with charged deflection plates can resul
144. de access to the bottom of the injection chamber 8 Pry out the O ring using a small flat blade screwdriver or a pair of tweezers Take care not to damage the O ring if removing it for cleaning or the O ring groove Q Withdraw the O ring When cleaning the O ring use a soft laboratory use cloth sprayed with ethanol and then wipe dry Also clean the groove in the sample injection chamber and the top surface and circumference of the sample tube agitation unit that forms the seal with the O ring Changing the Sample Loader O Ring soueUuslUle 9g Jaideyy I 161 soueuajUley g sajdeyuy 162 1 0 Insert a new or cleaned O ring into the O ring groove and run your finger around the O ring to ensure it is seated properly 11 Lower the sample loader door and reinsert the sample probe 12 Turn on the compressed air supply 13 Close the flip up door 14 Turn on the main power supply The sample loader waste catcher will pop back out by itself Cleaning the Sheath Tank and Sheath Line Cleaning the Sheath Tank and Sheath Line The sheath tank and sheath line should be cleaned regularly with ethanol to suppress the growth of bacteria Precautions when cleaning with ethanol Ensure the laboratory is well ventilated during cleaning with ethanol Inhaling ethanol vapor can cause irritation of the eyes skin and respiratory tract loss of coordination drowsiness and in sufficient concentration u
145. ders adapters may freeze if immediately placed on the collection stage when removed from the refrigerator For details about starting the SHSOOO instrument and SHSOO Software see System Startup page 41 and Logging In page 42 Click the m dialog launcher in the Temperature Control group on the Cytometer tab of the ribbon The Temperature Control Settings dialog appears 2 Select the temperature setting for the sample fluid then click Close Temperature Control Settings Sample 3 Ea C Collection 5 virc Unit 6l lS Sample 5 C 41 F or 37 C 98 6 F Collection 5 C 41 F only When sorting with the temperature set to 5 C 41 F place the collection tube holder in a refrigerator to cool the holder before loading it on the collection stage 3 Totuma temperature control device on off click Sample or Collection in the Temperature Control group on the Cytometer tab of the ribbon The buttons toggle between on and off If the temperature control function is turned off expected acquisition results may not be obtained depending on the sample being analyzed Precautions when temperature control is set to 5 C 41 F e Cool the collection holder to approximately 5 C 41 F in a refrigerator before loading it onto the collection stage Cooling the collection holder beforehand significantly reduces the time required to cool the collection tubes e While the t
146. e 813 deyo I 153 eoueuajUleyy g Jajdeyy 154 Droplet camera window Laser windows Collection stage and collection tube holders Clean the collection stage and collection tube holders carefully especially the upper portion of the collection stage where the unit is most exposed to salts and biological matter Transparent protective covers in the sorting area Wipe the inner surfaces of the transparent protective Covers Moisten a clean soft lint free cloth with water and wipe the covers General Cleaning Laser windows Transparent protective covers Wiping with a dry cloth can generate static electricity which may adversely affect the side streams during auto calibration Never use a dry cloth Sample loader Clean carefully to remove all salt products Spray a clean soft lint free laboratory use cloth with ethanol 70 from a spray bottle and wipe with the cloth Moisten a clean soft lint free laboratory use cloth with clean water and wipe the sample tube loader Do not operate any hardware or software controls while cleaning the sample loader or the sorting area When cleaning the sample loader remove the sample tube holder and clean separately When cleaning the sorting area pull the collection stage out to its maximum travel and remove the collection tube holder and clean separately When cleaning the sorting area also remove the deflection plates page 155 and wa
147. e data acquisition will stop automatically when the number of detected events exceeds 100 000 000 or the elapsed time exceeds 12 00 00 whichever occurs first e Recording stops data acquisition automatically when the recording stop condition is satisfied e Sorting stops data acquisition automatically when the sorting stop condition is satisfied e Recording amp Sorting stops data acquisition automatically when both the recording and sorting stop conditions are satisfied 5 Configure the detector settings Adjust the gain threshold and other settings For details see Configuring Detector Settings page 79 6 Specify the recording stop condition in Stop Condition under Recording You can select None Event Count or Elapsed Time for the recording stop condition and select a value from the drop down list You can also enter a value directly from the keyboard Recording Elapsed Time 00 00 00 Event Count 0 100 900 C Stop Condition Event Count 100 000 7 1 7 Click Start B Sample Group 1 iy E Tube 1 Status Pause Elapsed Time Total Event Event Rate 0 eps b gt Restart Stat Stop Record Sample Stop Condition None Y Sample fluid starts flowing from the sample tube into the sorting chip and data acquisition starts Acquiring Data 49 uoneiedo oiseg g13 deyo Kii uoleiado olseg Ja deyy 8 Click Record D
148. e user in the area on the right The Edit Account dialog appears 2 Edit the settings then click OK Edit Account Username liseri Full Name Organization Accessibility For details see step 4 page 38 in Adding Users The account settings are updated Changing Passwords Individual users can change their password used to log in to SH800 Software 1 Click the File tab then click Information in the menu on the left 2 Click Change Password rS Institution Information Show your institution informat ion Editable by administrator only The Change Password dialog appears 3 Set each field then click OK Change Password Username useri Old Password New Password Confirm Password OK Cancel Old Password Enter the current password required item New Password Enter a new password comprising up to 20 alphanumeric characters required item Confirm Password Enter the same password again for confirmation required item The password is updated Configuring Users 39 uoneiedaig guaideyy HIN uoleisdo olseg Ja deuyD 40 Basic Operation Chapter This chapter describes the basic operating procedure for running a simple experiment acquiring and analyzing data and sorting selected populations in the sample Workflow Workflow Start the system page 41 Turn on the power to start the SH800 Log in page 42 Setu
149. e Initial Instrument Setup screen or using Sample line exchange in maintenance mode For details see Logging In page 42 and Running Maintenance Mode page 158 3 Open the flip up door on the front panel ey 4 Remove the sample line from the pinch valve then click Next To remove grasp the sample line on both sides of the pinch valve and pull the sample line out towards you Pinch valve 5 Grasp the T shaped portion of the sample line connector and gently unscrew it counterclockwise to remove it from the inlet port on the chip loader then click Next soueUuslUle g Jaideyy IIN Changing the PEEK Sample Line PEEK sample line compatible models 1 69 eoueuajUleyy g Jajdeyy 170 Take care when removing the sample line Sample fluid may be partially conductive or highly corrosive which can damage electronic circuits 1f any droplets fall down between the gaps in the sample loader cover and come into contact with internal components Grasp the black fitting on the PEEK sample line and gently unscrew it counterclockwise to remove it then click Next e Unscrew the black fitting while holding the probe adapter down e If the sample probe cannot be pulled out the black fitting may be turning in the reverse direction due to twists in the sample line Unscrew slowly without releasing the black fitting e Take care when handling the sample probe It is a high precision comp
150. e compatible models page 169 or Changing the Sample Line and Probe Non PEEK sample line compatible models page 172 Initial Instrument Setup Load a sorting chip select the lasers to be used and insert the optical filters in the required optical filter pattern by following the on screen instructions in the Initial Instrument Setup wizard Hold the QR code on the sorting chip packaging in front of the camera on the host computer QR code SONY Sorting Chip for Cell Sorter SH800 m T Nozzle Size 100 micron Part J82aR1N 000000000 LLLL SSSS Research Use Only T YYYY MM DD The camera automatically scans the QR code Tip You can click Abort to bypass instrument configuration and proceed directly to the main window In this mode SH800 Software can be used only for analysis of existing recorded data in experiments When loading a sorting chip the QR code on the chip packaging must be scanned using the built in camera on the host computer to uniquely identify the chip and register the serial number and nozzle size of the chip with the SH800 This function maintains a history for the chip in order to ensure optimum performance When the QR code is successfully scanned the part number and nozzle size are displayed together with basic instrument settings The serial number for the chip is registered internally When the sorting chip is scanned successfully the serial number and o
151. e group templates and recent sample groups that can be used as templates You can search the template list by keywords Public Templates Displays sample group templates that were created as shared templates Blank Template An empty template My Templates Displays private sample group templates that you have created Recent Sample Groups Displays recent sample groups that can be used as templates Sample group structure Displays the internal structure of the sample group template selected in the list Sample group settings Displays configurable information and instrument settings for the sample group You can also edit settings after an experiment has been created For details about configurations items see Experiment settings page 111 in the Create Experiment Window Add Sample Group Adds a new sample group with the specified settings to the experiment structure Add Tube dialog The Add Tube dialog is displayed by clicking Add Tube in the Create Experiment window The Add Tube dialog is used to add a sample tube to the selected sample group in the experiment structure 2 Tube Templates list Displays the available tube templates and sample tubes that can be used as templates You can search the template list by keywords Public Templates Displays sample tube templates that were created as shared templates Blank Template An empty template My T
152. e main unit and insert the filter bypass line 13 In SH800 Software click Sheath Filter in the De bubble group on the Cytometer tab of the ribbon A popup window appears Cleaning the Sheath Tank and Sheath Line 14 Click Start to begin cleaning then click Close when finished 1 5 Click Ethanol Cleaning in the Shutdown group on the Cytometer tab of the ribbon Follow the on screen instructions to clean the instrument with ethanol When cleaning is completed both the main unit and SH800 Software automatically shut down A prompt to reconnect the sheath filter appears on the Ethanol Cleaning screen However when cleaning the sheath tank and sheath line leave the bypass line in place and proceed to the next step 16 Dispose of the ethanol in the tank using the same procedure in steps 5 and 6 Dispose of the ethanol in accordance with the laboratory rules and local ordinances and regulations 17 Pour 4 liters 1 1 gallons US of DI water into the sheath tank and reattach the lid e Do not touch the inner surface of the lid of the sheath tank Also take care not to spill any DI water e Do not use a pump or other object that has not been sterilized for pouring DI water into the tank e When inserting the sheath tank lid make sure that the black rubber seal is attached to the lid 18 Stow the sheath tank in the tray using the same procedure as in steps 9 and 10 1 9 Turn on the POWER STANDBY
153. e software DirectShow NET Source codes can be obtained from the Others OSS SourceCode folder on the supplied installation disc However Sony cannot accept inquiries regarding the content of these source codes For license information refer to the Others License folder on the supplied installation disc Apache License This product uses the Apache license software ZXing For license information refer to the Others License folder on the supplied installation disc Microsoft Public License Ms PL This product uses the Microsoft Public License Ms PL software WPF Toolkit DotNetZip and Fluent Ribbon Control Suite For license information refer to the Others License folder on the supplied installation disc Optional Accessories Software License sno ueLjj v s y g xlpueddy 225 sno ugjj siy g xipueddy 226 Environmental Notices Disposal of Old Electrical amp Electronic Equipment Applicable in the European Union and other European countries with separate collection systems This symbol on the product or on its packaging indicates that this product shall not be treated as household waste Instead it shall be handed over to the applicable collection point for the recycling of electrical and electronic equipment By ensuring this product is disposed of correctly you will help prevent potential negative consequences for the environment and human health w
154. e the collection stage is moving Select the appropriate plate sorting method in Sorting Method Always select the sorting method matching the collection tube device loaded on the collection stage w Sort Control v TA Unload Collection Sorting Method amp Well Strips gaa Sort Settings The sorting parameters vary depending on the selected sorting method Click Sort Settings x Sort Control TAN Unload Collection Sorting Method amp Well Strips F saa Sort Settings The Sort Settings dialog appears Configure the settings for sorting into wells on the Plate Sort Settings tab The description in this section shows an 8 well strip as an example The operation is the same for glass slides Sorting Sort Settings 8 Well Strips 0000000 88000008 E Q If using index sorting enable the Add index sort information checkbox In index sorting you can associate the cells sorted in each well with the data points for those cells on plots For details see Index Sorting page 101 2 Select the target well to set You can select the well using the following buttons or by clicking the well on the palette I Select wells in alternate columns Select wells in alternate rows Select wells in groups of four You can select multiple wells using the Shift and Ctrl keys The ID is displayed in Sort ID You can change the value in Sort ID
155. e the sheath pressure Increasing the sheath pressure reduces the laser excitation energy received and the intensity of the resulting fluorescence signal but is effective for increasing the sample throughput rate Hydrodynamic Focusing Sample fluid and sheath fluid are forced into the sorting chip using air pressure The sample fluid is injected into the center of the sheath fluid stream hydrodynamically focusing the sample fluid within the sheath fluid according to the principles of fluid dynamics The internal micro channel structure of the sorting chip encases the sample fluid within the sheath fluid to ensure high sensitivity stable optical detection characteristics Sample fluid Sheath fluid Sheath fluid IS 4 bos v l Y Sample fluid core lt Y lt E To nozzle The relative difference in pressure between the sheath fluid flow and the sample fluid flow forces the diameter of the sample fluid core to vary When the sample pressure is lowered the diameter of the sample fluid core decreases focusing the sample fluid into a narrow core stream When the sample pressure is raised the diameter increases with a corresponding increase in the sample event detection rate Ww V Low sample 2S High sample flow rate flow rate The flow of sample cells encased in sheath fluid passes the optical detection point where the fluid core is interrogated by up to four lasers of different wa
156. e worksheet The current zoom factor is displayed on the button icon This setting 1s applied to all plots Click this button and select the zoom factor from the menu or use the slider to set the zoom factor W o TBc 109 Favorite Cc Settings Pic Favorite Settings Allows you to store favorite worksheet settings e Save as Favorite Saves the current worksheet state as the default settings e Restore from Favorite Applies saved settings to the current worksheet Tools group Contains tool buttons for copying exporting and displaying data on the worksheet co amp amp o a Copy Worksheet Expor Data Show Picture to CSV Fie Magnifier roots Copy Worksheet Picture Copies the selected worksheet to the clipboard for pasting in other software for example to produce reports Export Data to CSV File Exports the recorded data for the sample tube as a CSV file Clicking this button displays the Exporting Data dialog page 128 If conducting index sorting you can export only the index sorted data by selecting Well Select Mode in the Index Analysis dialog page 102 Show Magnifier Shows hides a worksheet magnifier You can also set the zoom factor l Show Analyze Magniner index Data 200M Factor Index Sorting group Contains a command button for analyzing data using the index sorting function i Analyze index Data Li m Analyze Ind
157. eSingle Cell 95 Sorting area Door 16 Door button 16 Sorting chip 12 25 Alignment 46 Exchanging 67 Loading 43 Index 22 con Ill 228 QR code 43 thumbwheel 185 Sorting chip thumbwheel 17 Sorting mode 93 Normal 96 Purity 97 Semi Purity 96 Semi Yield 98 Ultra Purity 97 Ultra Yield 99 Yield 98 Starting 41 System components 10 T Troubleshooting 215 U Users adding 38 W Waste catcher cleaning 157 O ring 158 Waste tank 23 emptying or replacing 148 Wavelength laser 201 Window extension 199 Workflow 40 Index The material contained in this manual consists of information that is the property of Sony Corporation and is intended solely for use by the purchasers of the equipment described in this manual Sony Corporation expressly prohibits the duplication of any portion of this manual or the use thereof for any purpose other than the operation or maintenance of the equipment described in this manual without the express written permission of Sony Corporation Trademarks e Microsoft and Windows are registered trademarks of Microsoft Corporation in the United States and or other countries Intel and Intel Core are trademarks or registered trademarks of Intel Corporation or its subsidiaries in the United States and other countries e QR Code is a registered trademark of DENSO WAVE INCORPORATED URL http Awww qrcode com faqpatent html Alexa Fluor Qdot and PE Texas Red are registered trademark
158. ed This time is called the dead time These events are aborted automatically and cannot be sorted reducing the potential yield From this discussion it can be seen that sorting only a single targeted population where the targeted events occur in the center of droplets that contain no other unwanted events will always produce the best sort results in terms of purity Sorting all events for a single targeted population will always produce the best results in terms of yield Single Cell Mode In Single Cell mode only droplets containing a single targeted cell within the center region of the cell are sorted adjacent droplets are also sorted when using a 130 um sorting chip if the adjacent droplets are empty If an adjacent droplet contains the same type of target cell the droplet is not sorted 100 um sorting chip A droplet is sorted if it contains a single targeted event located within the center region of the droplet and both of the adjacent droplets are empty One droplet is sorted for each event matching the sorting criteria Not sorted Target lt 1 cell is outside L lt center of droplet 130 um sorting chip A droplet is sorted 1f it contains a single targeted event and the adjacent and subsequent droplets on both sides are all empty Three droplets are sorted for each event matching the sorting criteria P DS Not sorted Target cells are in adjacent droplets Sorted Single cell is i
159. elease catch to withdraw the connector Do not touch the surfaces of the ethanol connector that come into contact with ethanol Take care not to spill any fluid Remove the ethanol tank from the fluidics cart for easy access to the cap of the tank Remove the cap and tank probe from the ethanol tank and place in a clean safe location Refill the ethanol tank with the specified volume of ethanol and reattach the cap Place the tank in the fluidics cart tray connect the ethanol line and stow the tray Insert the line connector and push until it locks into place Carefully clean any residue fluid from the connector and outside surfaces of the tank Cleaning the Sample Fluidics System using Bleach Cleaning the Sample Fluidics System using Bleach The sample line and probe should be cleaned regularly using a protein dissolving bleach to disinfect the lines that carry the sample fluid The fluidics system should be disinfected on a regular basis or as required especially if running samples that contain for example infectious substances The sorting chip PEEK sample line sample line and sample probe can be disinfected using a 1 to 3 Sodium Hypochlorite bleach solution Cleaning the sample probe using a high concentration bleach solution 5 or higher may cause rusting or discoloration Use normal concentration aqueous solution 1 In SH800 Software click Bleach Cleaning on the Cytometer tab of the rib
160. emperature is set to 5 C 41 F do not remove the collection tube holder from the collection stage Insert and remove the collection tubes directly from the collection tube holder Loading a collection tube holder that is warm or that has become warm may result in a sudden change in temperature perhaps initiating a temperature control error causing the main unit to be reset When unloading and loading the collection tube holder temporarily turn off the temperature control function wait about 30 seconds and then set the temperature to 5 C 41 F again The metallic parts of the 96 well plate holder become cold when the holder is cooled Always load and unload by holding the plastic sides Preparing Collection Tubes Prepare the collection tube device for collecting droplets containing target cells for sorting The SH800 supports the following collection tube devices Each type of collection tube is used with a corresponding holder that mounts on the collection stage Collection tubes multi well plates 15 ml conical tubes Collection tube holder 15 ml 5 ml round tubes Collection tube holder 5 ml 8 well strips 8 well strips holder Slide glass Slide glass holder 6 well plates 96 well plate holder 12 well plates 24 well plates 48 well plates 96 well plates 8 well PCR strips 96 well PCR plates 384 well plates 96 well PCR plate holder 384 well plate holder 384 well PCR plates 384 well
161. emplates Displays private tube templates that you have created Recent Tubes Displays recent sample tubes that can be used as templates Sample tube structure Displays the internal structure of the sample tube template selected in the list Sample tube settings Displays configurable information and instrument settings for the sample tube You can also edit settings after an experiment has been created For details about configurations items see Experiment settings page 111 in the Create Experiment Window Worksheet Displays the plots and gate statistics configured for the selected template or sample tube Add Tube Adds a new sample tube with the specified settings to the selected sample group in the experiment structure Main Window The main window is where you acquire and record data perform analysis run cell sorting and other functions The main window has the following configuration Ribbon e File tab page 104 e Experiment tab page 113 e Cytometer tab page 117 e Compensation tab page 124 e Worksheet Tools tab page 125 Plot Tools tab page 129 e Gate Tools tab page 130 Worksheet Gates and Statistics table page 131 context menu page 139 Acquisition tab page 132 Experiments tab page 133 context menu page 139 Sorting control pane page 136 Experiment Tab ribbon The Expe
162. ence cannot be aborted once it is started e The SH800 hardware and software are automatically shut down after cleaning with ethanol You can also run ethanol cleaning in maintenance mode For details see Running Maintenance Mode page 158 2 Click Start to begin the cleaning process and follow the on screen instructions 3 Refill the DI water tank in the main unit with distilled deionized water then click Next Make sure that there is sufficient DI water in the tank An alarm will be raised if there is insufficient volume of DI water in the DI water tank before cleaning See Refilling the DI Water Tank page 149 4 Refill the ethanol tank in the fluidics cart with ethanol then click Next Make sure that there is at least 0 5 liters 18 fl oz in the ethanol tank An alarm will be raised 1f there is insufficient volume of ethanol in the ethanol tank before cleaning See Refilling the Ethanol Tank page 151 5 Remove the sheath filter Q Remove the sheath filter from the retaining clamp without disconnecting the connectors 2 Disconnect the sheath filter lines from the UPPER and LOWER connectors Press the metal release catch and withdraw each connector The sheath filter connectors have stop valves that prevent leakage of fluid when disconnected Cleaning the Internal Sheath Line and DI Water Line using Ethanol soueuslUle g Jaydeyy I 189 soueUslU
163. epare automatic setup beads in advance if planning to run automatic calibration Filter the automatic setup beads using a 30 um filter 2 Insert 1 ml or more of undiluted automatic setup beads in a sample tube and place the tube in the corresponding sample tube holder Always use automatic setup beads in undiluted form Sample Temperature Control The SH800 is fitted with thermal control units in the sample loader and collection stage to maintain sample fluid temperature control while running an experiment The temperature control device provides basic control of temperature and can be set to 5 C 41 F or 37 C 98 6 F You configure settings in SH800 Software e The temperature control function in SH800 Software cannot be used to cool 8 well strip holders slide glass holders 96 well plate holders 384 well plate holders or 384 well PCR adapters However the holders contain an embedded cooling agent You can cool a holder in a laboratory refrigerator 20 C 4 F beforehand and then place the holder on the collection stage For 8 well strip holders slide glass holders and 96 well plate holders Preparing a Sample Cooling time 5 hours or longer Temperature retention time About 1 hour varies with ambient temperature For 384 well plate holders and 384 well PCR adapters Cooling time 8 hours or longer Temperature retention time About 1 hour varies with ambient temperature e Note that hol
164. er Operator Name of the operator Memo Descriptive comments OK Applies the settings and closes the dialog Cancel Cancels settings and closes the dialog Apply Applies the settings without closing the dialog Sample Group Information dialog The Sample Group Information dialog is displayed by double clicking Sample Group Information in an experiment in the Experiment Explorer The Sample Group Information dialog is used to view and edit information about the sample group Sample Group 1 Sample Group Information Sample Group name bample Group 1 Date 12 28 2013 9 47 14 PM Sample Group Name Unique name of the sample group required item Date Displays the date the sample group was created This parameter cannot be modified Species Name of the target biological species Cell type Name of the cell type within the biological species Memo Descriptive comments OK Applies the settings and closes the dialog Cancel Cancels settings and closes the dialog Apply Applies the settings without closing the dialog Tube Information dialog The Tube Information dialog is displayed by double clicking Tube Information for a sample tube in the Experiment Explorer The Tube Information dialog is used to view and edit information about the sample tube Tube 1 Tube Information a 3 E 3 3 ge ae wD Da f m m fi g IO za a i Tube Name Unique name
165. er 1 e 96 well PCR plate holder 1 SH800ZP options e 384 well plate holder 1 e 384 well PCR adapter 1 SH800 and SH800Z options e 8 well strips holder 1 e Slide glass holder 1 Documentation Structure The system documentation comprises the following manuals e Safety Guide Contains safety information usage precautions and specifications Read carefully before operating the unit e Operator s Guide Describes the required preparation and basic operating procedures in order to use the instrument adjustments and procedures for more advanced use of the instrument and the configuration items in the SH800 Software windows and dialogs Chapter 3 Basic Operation page 40 provides an overview of the basic procedures from setting up and calibrating the instrument through to acquiring data analyzing data and sorting Refer to the subsequent chapters for detailed information about operating procedures and instrument settings Components and Documentation 11 MAIMOAQ_ Je deyD 12 Overview Main Features The LE SH800 series is a benchtop high speed multilaser flow cytometer and cell sorter designed for research laboratory use It employs a novel replaceable microfluidics cell sorting chip that features high reliability and greatly simplifies cell sorting setup It can be easily and quickly exchanged as needed for quick turnaround between measurements to minimize system downtime an
166. er is invalid Shut down the cytometer and wait a while before trying The laser configuration is invalid again If the problem persists contact your Sony distributor for The ADC configuration is invalid service Power supply error on the ADC board Power supply error on the ADC2 board Timed out to close the sample door Timed out to control sheath pressure Sheath Fluid Tank is almost empty Refill the sheath tank See Refilling the Sheath Tank Refill the sheath fluid tank following the instructions in the page 146 operator s guide Waste Fluid Tank is almost full Replace the waste tank See Emptying Changing the Waste Empty the waste tank following the instructions in the Tank page 148 operator s guide Collection area door is open Close the sorting area door Close the collection area door The flip up door is open Close the flip up door Close the flip up door Warning for the 561 nm laser Laser output does not decrease immediately The laser can continue to be used but should be replaced as soon as possible Warning for the unsuccessful communication on MD2 board This warning does not affect automatic calibration operation although it may appear during automatic calibration In this case continue the automatic calibration process Warning for the unsuccessful communication on PCT board Shut down the cytometer and wait a while before trying again Warning for the unsuccessful comm
167. eriment group on the Experiment tab of the ribbon The Create Experiment window is used to create a new experiment using templates Add Sample Group Add Tube Experiment Templates list Displays the available experiment templates and experiments that can be used as templates You can search the template list by keywords Public Templates Displays experiment templates that were created as shared templates Blank Template An empty template My Templates Displays private experiment templates that you have created Recent Experiments Displays recent experiments that can be used as templates Experiment structure Displays the internal structure of the experiment template selected in the list You can add and delete sample groups and sample tubes as required Experiment settings Displays configurable information and instrument settings for the experiment You can also edit settings after an experiment has been created Experiment Information Specifies the name and other basic information of the experiment The date cannot be modified For details about each item see Experiment Information dialog page 134 Sample Group Information Specifies information relating to the sample groups For details about each item see Sample Group Information dialog page 134 Create Experiment Window uondos q mopuiM Z JeIdeyD I 111 uondiosaqg MOpUIN Z Je deUu
168. es and Statistics table displays the following information Name Displays the names of gates and parameters Events Displays the number of events Parent Displays the proportion of events of each gate relative to its parent Total Displays the proportion of events of each gate relative to the total of all events Other statistics values Displays the values of statistics configured in the Statistics Editor dialog e Mean Average value of events in population e Minimum Minimum value in a population e Maximum Maximum value in a population e SD Standard deviation of events from the mean value in a population e CV Coefficient of variation of events in a population e Median Median value equal number of events above and below Mode Value with highest event count corresponds to the highest peak on a histogram plot e HPCV Half peak coefficient of variation coefficient of variation from width of distribution at half peak height For details see Displaying Statistics page 92 Gate hierarchy Displays the hierarchical relationship between gates The color to the left of the gate name shows the color of the gates on plots Main Window uondos q mopuiM Z JeldeyyD I 131 uolduoseg mopuiM Z Je deuyD 132 Acquisition Tab control pane The Acquisition tab controls data acquisition and recording B Sample Group 1 ay r4 Tube
169. ese 108 Help Window succicthut cous teatro ahtee en aaiemeneienls 110 LOL IW GOW seie ENT 110 Create Experiment Window cccesssseeseesssseeeeeennens 111 Main VWI GOW ssscsavecsesicnsntewetstasndenvaneestecavsamenleubesnestunaiweewseats 113 Expenment Tab 11 BDO cnreccorinnnin 113 Cytometer Tab BODON sioi 117 Compensation Tab ribbon seossesssseseeesssssssssererrssssssssseees 124 Worksheet Tools Tab ribbon sssesssessessensenssessesssessessessees 125 LPIOE Cooh Lap iD DOM essan a 129 Gate Tools lab nibOOM eienn e obec ean 130 Gates and Statistics Table wo cc eecceesceeccesceesceeseees 131 Acquisition Tab control pane ccecceccesssseeeeeeeeeeeeeaeees 132 Experiments Tab control pane ou cceeseeeeeeeeeeeeeeeeeees 133 Sorun COMmol Pane 6 csacics teria siocieae ersten ee deaeneneeys 136 GONTEXE MENU puisessa aa 139 Experiment Explorer Context Menu sssssssssseeeeeeereeeessssssss 139 Worksheet Context Menuaren Giiaciniiuaceaeens 140 Chapter 8 Maintenance Maintenance Schedule ccccccceeseeceeseeeeeeeeeessesenseeeeees 143 Preparations for the Day Before Measurement 143 Maintenance for Each Startup Operation ccccccceeeeeees 143 Maintenance for Each Shutdown Operation ccc cceee 143 Monthly Maintenance oi 0 i sneaieciveswt ie nde dn teeter ands honest 144 Periodic Maintenance ic5 3ccoscedscct
170. etector FL1 is almost totally due to the spectra of fluorochrome A The output of detector FL3 is predominantly due to the spectra of fluorochrome B but it does contain a small component approximately 10 due to fluorochrome A However the output of FL2 contains significant components from the spectra of both fluorochromes A and B This problem increases considerably as the number of fluorochromes being simultaneously detected increases Intensity FL FL2 FL3 Emission Emission spectra of spectra of fluorochrome A fluorochrome B A Wavelength Fluorescence compensation is the process of calculating the components of the detected signals due to each fluorochrome in the sample and subtracting the unwanted components from each channel This converts the channel detector output from signals of fluorescence intensity at a given wavelength to signals of fluorescence intensity due to each fluorochrome Compensation adjusts the mean value of fluorescence intensity of populations that are not stained with a particular fluorochrome to the mean value of a negative control sample which by definition is not stained with any fluorochromes When compensated the populations effectively appear aligned into quadrants on 2D plots Negative for X Positive for Y Fluorochrome Y Positive for X Negative for Y Negative for both X and Y Fluorochrome X Optical System e The visual representation of the compensated data on a 2D plo
171. ex Data Displays the Index Analysis dialog for emphasizing the position on plots of cells sorted in each well in multi well plates For details about the Index Analysis dialog see Analyzing Index Sort Data page 102 Compensation group Contains tool buttons related to fluorescence compensation Do Apply Compensation Manual Compensation aie ee Se Apply compensation Selects whether fluorescence compensation is applied to plots on the worksheet Clicking this button toggles compensation on off Manual Compensation Toggles manual fluorescence compensation mode on off for the plots on the worksheet Print group Contains tool buttons for printing statistics and plots displayed on the worksheet ee Print Custom Print Print Print Displays the Print window page 108 for printing the worksheet Custom Print Displays the Custom Print dialog page 128 for selecting the items to print Gate Editor dialog The Gate Editor dialog is displayed by clicking Edit Gate on the Worksheet Tools tab Plot Tools tab or Gate Tools tab of the ribbon The Gate Editor dialog is used to edit gates e Details tab Edits attributes of gates Tube 2 Data Source 1 Gate Editor Main Window uondos q mopuiM Z JeIdeyD I 127 uolduoseg mopuiM Z Je deuyD 128 Gate List Displays a list of the configured gates You can show or hide a gate on plots
172. experiment Double clicking this component displays the Experiment Information dialog page 134 Sample group Displays the name of a sample group A sample group contains the following components Sample Group Information Contains information about the sample group Double clicking this component displays the Sample Group Information dialog page 134 Measurement Settings Contains instrument settings Double clicking this component displays the Measurement Settings dialog page 73 Compensation Settings Contains the fluorescence compensation settings for the fluorochromes used Double clicking this component displays the Compensation Settings dialog page 124 g Compensation Panel Contains one negative unstained control tube and up to six positive fluorochrome stained control tubes A compensation control tube is required for each fluorochrome used to mark samples in the sample group The amp icon indicates sample tubes for which data has been recorded or which have been used for sorting These tubes cannot be reassigned as the active tube Double clicking a tube displays the worksheet for the tube Negative control Tube for recording data for the negative control sample used to compensate for cellular autofluorescence amp Positive control tubes Tubes for recording single stained positive control samples used to calculate the fluorescence compensation spillover matrix one tube
173. experiment structure 4 Modify the experiment and sample group information as desired A Experiment Information Experiment 12 28 2013 8 36 01 PM 12 28 2013 8 36 01 PM i Sample Group Information Sampie Group 1 Tip The name of the experiment is supplied automatically although it is recommended that you change it to a more appropriate name Select the detector channel pulse parameters for data acquisition in the Measurement Settings pane For details see the Measurement Settings dialog description in Configuring an Experiment page 73 A Measurement Settings Parameter Settings Brilliant Violet 42 Y FITC v v PE v APC v Y PerCP Cy5 5 v i PE C v Y J 405 el FSC FSC 4 BSC 30 0 J ase 5 00 FLI 40 0 FL2 40 0 iB 561 FL3 40 0 FL4 400 E 638 FLS 40 0 FL6 40 0 P S e The fluorochrome and marker names are optional but recommended e Check that the fluorochrome and marker names assigned to detection channels correspond with the laser configuration and optical filter pattern For details see Fluorochrome Detection Matrix page 204 When finished building the experiment click Create New Experiment in the bottom right hand corner of the window Create New Experiment If Blank Template was selected the New Experiment Startup Procedure dialog page 48 appears If an existing template or experiment was selected the main window appears T
174. ext menu eye en eE ere A Te ee SU FST Te oF Sere Ss ee pL Be Create Density Plot 6 0 Events Create Histogram Plot D f te Create Dot Plot 40 rm 20 ch Move Color Order to Back Copy Region Ctri C Visible Gate Delete GS Remove Send to Back Set as Calculation Target F4 Properties 0 ii 1 Briliant Violet 421 4 2 Click Calculate Matrix on the Compensation tab of the ribbon Fe Experiment Cytometer Compensation maan PNA Compensation Show Wizard The Calculate Compensation Settings dialog appears 3 Check that the specified gate is the target of the calculation and click Calculate to calculate the spillover matrix Calculate Compensation Settings gt Brilliant Violet 421 es 2 i mc es Briliant Violet 421 10000 000 000 090 0 00 10000 000 000 ooo 0 00 10000 0 00 0 00 000 0 00 100 00 6375 16 4996 96 421645 41033 100 00 644 43 000 000 000 000 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 100 00 A Click Close to close the dialog Adjusting Fluorescence Compensation Manually You can modify the compensation settings after using the Compensation Wizard or you can enter fluorescence compensation values manually to edit the spillover matrix directly e You can save the current fluorescence compensation settings using the Compensation Settings dialog For details about di
175. f the filter facing up Changing the DI Water Filter 6 Connect the two fluidics lines from the DI water filter to the corresponding connectors Connect the line from the top of the filter to the UPPER connector and the line from the bottom to the LOWER connector Insert each connector and push it until it locks into place A click sound occurs when the connectors lock into place Place the DI water filter in the retaining clamp 8 Close the fluidics maintenance door and turn on the power supply and log in 9 Open the fluidics maintenance door remove the DI water filter from the retaining clamp without disconnecting the connectors and check the fluid level 1 0 Turn on the power supply to the main unit and start SH800 Software 1 1 Click Settings on the Cytometer tab of the ribbon to display the Cytometer Settings dialog Click DI Filter de bubble on the Maintenance tab The DI Filter de bubble dialog appears 12 Click Start 13 Release the air trapped in the DI water filter Slowly open the air release port on the DI water filter within five minutes of startup to release the trapped air If air is not released within five minutes after startup turn the power supply to the main unit off and then on again then repeat the procedure e It is recommended that the drip tray be pulled out partway before proceeding Slowly open the air release port while holding a soft clean c
176. fluid The sort delay is optimized during auto calibration Sort Phase The sort phase setting adjusts the phase difference of the charging pulse waveform as cells pass between the optical detection point and the breakoff point It is used to determine the timing of the electrostatic charging pulse applied to the sheath fluid The sort phase is optimized during auto calibration Correction The correction setting adjusts the shape of the charging waveform applied to successive droplets e Top spinbox Controls the overall charging waveform correction adjustment e Lower spinboxes Fine tunes the correction applied to the first droplet second droplet and third droplet respectively When droplets are electrostatically charged the next droplet receives an induced charge of the opposite polarity which can affect the angle of deflection as the droplets pass through the deflection plates a problem called fanning The correction function uses a defanning algorithm to adjust the charging waveform applied to successive droplets to reduce fanning Calibration Display Mode The calibration display mode changes the image displayed by the droplet camera so that only droplets containing automatic setup beads are displayed This mode is used for fine manual control of the sort delay sort phase and related sort parameters Save Droplet Info Saves the current droplet image as a reference image used by the Cont
177. for each fluorochrome used to mark samples Main Window uondos q mopuiM Z JeIdeyD I 133 uondosaqg mopuiM Z Je deuyD 134 Sample tube Tubes containing sample for data acquisition recording and cell sorting Each tube also has worksheet settings and sort amp stop settings associated with it Double clicking a tube displays the worksheet for the tube To make a tube the active tube for data acquisition select the tube and click Assign Tube in the Target group on the Experiment tab of the ribbon During acquisition the tube icon changes to The icon indicates sample tubes for which data has been recorded or which have been used for sorting These tubes cannot be reassigned as the active tube The following icons are displayed for each tube on the right side in the Experiment Explorer Indicates data is being recorded Indicates data has been recorded yy Indicates tube is being sorted w Indicates that the tube has been used for sorting into the collection tubes Tube Information Contains information about the tube Double clicking this component displays the Tube Information dialog page 135 Worksheet Settings Contains information about the plots gates and statistics on the worksheet Double clicking this component displays the Worksheet Settings dialog page 135 Stop amp Sort Settings Contains the sort settings and the stop conditions
178. g Scale Ranges To adjust a scale range automatically Select a plot then click one of the automatic scale adjustment buttons in the Axes group on the Plot Tools tab of the ribbon A hest Tools Plot Toots SH800 Softw Well Data Source 1 All Events A Rectangle Adjust Auto Adjust XY Automatically adjusts the scale of the X axis and Y axis Auto Adjust X Axis Automatically adjusts the scale of the X axis only Auto Adjust Y Axis Automatically adjusts the scale of the Y axis only To specify scale ranges manually Select a plot then click the la dialog launcher in the Axes group on the Plot Tools tab of the ribbon SH800 Softw hest Tools Well Data Source 1 Plot Toots Gate Tools All Events A iy Be e Lk fa 29 O Swap Auto Adjust Auto Adjust Auto Adjust Zoom Default Rectangle XY X Axis Y Axis Adjust Axe The Property Window dialog appears 2 Enter values for the minimum and maximum values for the X axis and Y axis in Axes LT Property Window Plot Properties Gate Name ME Paictte 61 All Events Gate Color Show Population Axes X Axis Y Axis Close 3 Click Close to close the dialog To zoom in using a mouse You can enlarge a portion of the plot you want to display using the mouse 1 Select a plot then click Zoom in the Axes group on the Plot Tools tab of the ribbon SH800 Softw hest Tool
179. g fluidics cart maintenance The cart can be locked unlocked using a coin or similar flat object Fluidics Cart vuLeuguIeN 813 deyo I 145 uLeu uIeN g sajdeyy 146 Refilling the Sheath Tank The sheath tank should be checked every time before sorting and refilled if it is close to empty Also the tank must be refilled when prompted to do so in SH800 Software e Sheath fluid is a sterile solution Avoid contact with the contents of the tank to maintain sheath fluid sterility e Never use the supplied sheath tank for any other purpose e When opening the sheath tank cap check that Standby is displayed on the LCD monitor or that the power to the main unit is turned off If Standby is not displayed place the unit in Standby mode using the following procedure G Click Settings on the Cytometer tab of the ribbon The Cytometer Settings dialog appears 2 Click Advanced Settings The Advanced Settings dialog appears 3 Click Standby on the Pressure Options tab 4 Check that Standby is displayed on the LCD monitor Do not force the lid down or place objects on the lid when the tank is pressurized Store replacement sheath fluid in a room with the same temperature environment as the instrument or allow the sheath fluid to acclimatize in the same room as the instrument before refilling the sheath tank Droplet formation may become unstable if the sheath fluid added to the tank is a
180. garithmic scale after fluorescence compensation has been applied to a tube The data transformation used during fluorescence compensation inherently introduces further variance into the measurement data due to variations in detector sensitivity background noise and other factors The result is that some data values can become close to zero or even negative When values close to zero are plotted on a log scale they are wedged up against the axis making them very difficult to discern In addition values below zero cannot be displayed at all This can lead to these events which are real events being excluded from gates or other analysis It can also lead to inaccurate fluorescence compensation when attempting to adjust compensation manually using visual cues Editing Plots In these cases a biexponential scale can be used to avoid some of the pitfalls of logarithmic scales while maintaining the overall utility of logarithmic scales Changing the Axis Parameters Click the axis title and select the desired parameter from the popup menu Tube 1 PerCP Cy5 5 PerCP Cy5 5 al All Events BSC A Brilliant Violet 421 FITC A PE A APC A PerCP Cy5 5 4 PE Cy7 A Height FSC H BSC H Brilliant Violet 421 4 FITC H PE H APC H PerCP Cy5 5 H PE Cy7 H Time Load Collection TIME seconds Events Sorting Methoil To Sort Events f Atada A preview of the plot appears as the mouse hovers over each Adjustin
181. gnostics are run to test the proper working condition of all subsystems The SH800 status appears on the LCD monitor Sheath Pressure Sheath Waste 20 00 psi Ethanol DI Sample Pressure 20 70 psi System Startup 41 uoneiedo oiseg g13 deyo Kii uo eiado oiseg Ja deuyD Logging In Start SH800 Software and log in to the SH800 Only one user has permission to operate the SH800 at any one time Other users may log in to monitor the operation but they do not have permission to operate the SH800 1 Select SH8000 Software on the Start screen to start the software The login window appears 2 Check that Standby status is displayed on the LCD monitor 3 Enter your user name and password then click Login Cell Sorter SH800 sename Password Tip The administrator account user name and password are both set to administrator when the unit is shipped It is recommended that you change the password for the administrator account for security considerations If login is successful the Initial Instrument Setup wizard appears initial Instrument Setup 1 Chip Detection Please scan the QR code printed on the Sorting Chip package Logging In Tip To change the sample line before initial instrument setup click Sample line exchange and follow the instructions in the on screen wizard For details see Changing the PEEK Sample Line PEEK sample lin
182. gs Displays the sort settings Instrument Setting Displays the instrument settings Auto Parameters Displays automatically configured sorting parameters Data Source Results dialog The Data Source Results dialog is displayed by selecting a data source in the Experiment Explorer and clicking Show Results in the Settings and Information group in the Experiment tab of the ribbon The Data Source Results dialog allows you to view the results of each data source The Data Source Results dialog displays all the information from the Tube Results dialog except the Basic Information tab For details see Tube Results dialog page 115 FCS File Export dialog The FCS File Export dialog is displayed by selecting an experiment in the Experiment Explorer and clicking Export to FCS file in the Export group on the Experiment tab of the ribbon or by right clicking and selecting Export FCS Files from the context menu O FCS File Export Select target tubes Selects the sample tubes to export Place a check mark in the checkbox for each target tube FCS Version Selects the FCS file version 3 0 or 3 1 Convert BSC to SSC Selects whether to change the sensor name for data recorded by the BSC back scatter detector to SSC side scatter in the exported FCS files Optional FCS Text Segment Keywords You can specify optional FCS text segment
183. h a cloth that is too dry This charge can adversely affect the side stream position causing an error in the automatic side stream calibration process If this occurs wipe the areas around the deflection plates with a cloth soaked in DI water to dissipate any accumulated electrostatic charge e Make sure the waste fluid outlet and O ring of the waste catcher are correctly aligned with the port on the mounting plate and the waste catcher is securely fastened to the mounting plate There is a risk of waste fluid leakage if not fitted correctly especially the O ring or risk of overflow if the port is obstructed e The O ring on the rear of the waste catcher must be replaced every 12 months e Check that the waste catcher screw is not wet when tightening the screw A malfunction may occur if the screw is wet when installed O ring Running Maintenance Mode Running Maintenance Mode The following maintenance can be performed in maintenance mode e Ethanol cleaning e Waste A maintenance bleach cleaning e Changing the sample line You can run maintenance mode from the SH800 Software login screen Running ethanol cleaning Precautions when cleaning with ethanol e Ensure the laboratory is well ventilated during cleaning with ethanol Inhaling ethanol vapor can cause irritation of the eyes skin and respiratory tract loss of coordination drowsiness and in sufficient concentration unconsciousness e Never place a
184. he automatic calibration sequence starts A progress bar displays the status at each stage of the calibration process Auto Calibration 2d A ae eel pedi A sad Droplet Clock 33500Hz 4 0 00 de Stream Calibratio 30 200deg o T r ADA EVLE elechite w w e i 0 0 oe D 0 X Position 0 D 429496 ZPosition 0 9 Wait 30 seconds for stream to stabilize As automatic calibration proceeds a histogram for the automatic setup beads the droplet camera image and the side stream monitor image are displayed Tip During automatic calibration you can click Abort to return to the previous screen When automatic calibration finishes Abort is disabled and Retry and OK are enabled Clicking Retry restarts the automatic calibration procedure 5 When calibration finishes click OK The Create Experiment window appears for building a new experiment During automatic calibration precision measurement 1s performed using the side stream monitor Accordingly do not place any bright light sources halogen lamps for example within the following angular ranges Doing so may cause automatic calibration to malfunction Top view PF a a oC Side view 13 E an mm Creating an Experiment When calibration finishes you create an experiment that is used to acquire data from samples in the Create Experiment window Creating a New Experiment This section describes how to create a new experiment
185. he created experiment is automatically made the active experiment and the first tube is automatically assigned as the active tube To add a sample group Use the following procedure to add a sample group to an experiment Click Add Sample Group in the Create Experiment window Add Sample Group Add Tube The Add Sample Group dialog appears Public Templates Measurement Settings COR ARLY A ti i al NEN ee 3 UR 2 UENEN 3 Creating a Customized Experiment 71 sluewuedxy Buunbyuoy p Jaideyo iii s yu wn dx Buunbyuog p Je deyD 2 Select a sample group template or recent sample group in Sample Group Templates SampleGroup Templates Public Templates ca Blank Template My Templates Recent Sample Groups Top 10 ai Group Sar iii roup mes Group Sa meee roup Group Sa mpe up SampleGroup Sa iii 1 Enter a name for the sample group and modify other settings as required The name of the sample group is a required item Sample Group Information Sample Group Select the detector channel pulse parameters for data acquisition in the Measurement Settings pane i Measurement Settings S IN S ISi WN IS gt v a 4 4 4 a 4 4 BSC 30 0 FLL 40 0 FL2 40 0 rm Hal D x 3 WN a 5 S b SES a Ain b MA wna so bi co fo wn Q gt A m N T a A so o D FL5 40 0 FL6 40 0 Only control tubes tha
186. he positions of only the cells in the selected gate on plots Only wells containing index sorting events encompassed by the selected gate are displayed Indek Analysis Analysis Mode Gate Select Moge v Selected Gates All Events Tip Clicking amp displays the User Preference dialog page 105 for setting user default preferences Adjusting Sort Parameters Manually Experienced Users During startup the SH800 automatically measures and calibrates the sorting parameters for sorting using automatic setup beads However there may be cases where you wish to override the automatic control system and adjust the sorting parameters manually These parameters are for experienced operators who are already familiar with manual sorting controls The manual controls are located in the Advanced Settings window 1 Click Settings on the Cytometer tab of the ribbon The Cytometer Settings dialog appears 2 Click Advanced Settings Cytometer Settings Control Chip Information Cytometer information Maintenance Laser Threshold i 405nm On Turn Off Channel FSC y e 7 mitt ere 7 a 48Enm On Turn Off Value 5 00 561nm O ff Turn On Sensor Gain FSC 4 85C 9 0 638nm Off Turn On X 4 4 a a FL 400 R L2 40 0 al F Temperature Control a z a S 5 or 3 40 0 40 09 lt Sample 5 vic FLS 0 0 m L4 40 0 3 5 FLS 40 0 6 40 0 Collection
187. he ribbon Exchanging the Sorting Chip 67 uoneiedo oiseg g13 deyo Kii uoleisdo olseg sa deuyD Cytometer Settings Control Chip Information Cytometer Information Maintenance Chip Type Sorting Chip Nozzle Size 100 um Part J82aR1iN Chip ID 00000000 0008 0005 Year Month Date 2012 01 10 Usage 405nm 2 hours 25 minutes 488 nm 9 hours 46 minutes 561nm 0 hours 0 minutes 638nm 0 hours 0 minutes Id 5 68 Backing Up Restoring the Database Backing Up Restoring the Database You can back up all data in the SH800 Software database You can also restore all data from a backed up database as required This operation is available only when logged in using an administrator account Tip e Backup restore operations are supported on the NTFS format file system Other format file systems are not supported e Backup operations are performed on external storage only Backing up the Database 1 Click Database on the File tab of the ribbon then click Backup Backup Backup the database Administrator Only D Restore Restore the database Ba Delete All Data ilt Clear the database Administrator Only The Backup Database dialog appears 2 Click Browse to specify the backup data destination then click Start Elapsed Time 00 00 00 Close The database backup commences and a progress bar is displayed When the database is backed up a confirmation message
188. he screen to clean the sample line When the fluidics check is completed the Auto Calibration screen appears Cleaning the Sample Line You can clean the sample line before running automatic calibration 1 Click Sample line cleaning on the Fluidics Check screen of the Initial Instrument Setup wizard Initial Instrument Setup v 1 Chip Detection Please check that sheath is dripping from the tip of sample probe If not click Sample line cleaning to start Bleach Cleaning and DI Rinse to clean sample line vV 2 Chip Information vV 3 Chip Loading Vv A Laser Setting n Vv 5 Filter Setting Fluidics check is in progress 6 Fluidics Check The Sample Line Cleaning wizard appears 2 Click Start to begin cleaning the sample line Sample Line Cleaning Click Start button to initiate Sample Line Cleaning Sample Line Cleaning will take about 10 minutes Warning DI Water Rinse cannot be skipped once Bleach Cleaning has been started Follow the on screen instructions In sample line cleaning the fluidics system is cleaned using a sodium hypochlorite solution bleach cleaning and DI water in that order e You can skip each cleaning step by clicking Skip but cleaning with DI water after cleaning with bleach cannot be skipped e You can also clean the system again after running the cleaning steps by clicking Cleaning again e For details about cleaning using Sodium Hypochlorite solu
189. he three fluidics lines sheath fluid waste fluid and ethanol and sheath air line from the rear of the fluidics cart Press the metal release catch to withdraw each connector e Do not touch the surfaces of the connectors that come into contact with fluids Take care not to spill any fluid e Always wear gloves and other protective clothing mask and goggles as required when handling waste fluid Disconnecting and Reconnecting the Fluidics Cart soueuslUle g Jaideyy I 187 soueUDslUle g Ja deuy 188 3 Disconnect the connection cable from the rear of the fluidics cart Unscrew the retaining screws and pull the connector out Cart connection cable 4 Move the fluidics cart or re route fluidics lines sheath air line and cable as required then reconnect the lines air line and cable Insert each connector and push until it locks into place 5 Turn on the power supply Check for any fluid spills and clean as necessary O Check the operation of the fluid level sensors in the main window in SH800 Software or on the LCD monitor Check the electrical cable connection 1f the status information does not appear Cleaning the Internal Sheath Line and DI Water Line using Ethanol Cleaning the Internal Sheath Line and DI Water Line using Ethanol The SH800 internal sheath line and DI water line should be cleaned with ethanol regularly to prevent the growth of bacteria and other agents
190. hich could otherwise be caused by inappropriate waste handling of this product The recycling of materials will help to conserve natural resources For more detailed information about recycling of this product please contact your local Civic Office your household waste disposal service or the shop where you purchased the product Environmental Notices Index Numerics 8 well strips collection 19 33 A AC power connector 22 Air filter tank 165 Analyzing data 83 Automatic calibration 46 B Back scatter BSC 196 Backup 68 Bandpass optical filter BPF 198 Bleach cleaning 152 C Cart connection cable 23 24 Chip insertion slot 17 Chip loader 17 Cleaning 153 Bleach 152 Ethanol 188 optical filter 186 Compensation introduction 199 Compressed air supply input connector 23 D Data analysis 51 83 Deflection plates cleaning 155 Detection matrix fluorochrome 204 Detection module 18 198 DI water filter autoclaving 183 De bubble 182 replacing 177 DI water tank 21 cleaning 166 refilling 149 DISPLAY MODE button 17 Drip tray 21 Droplet sorting 194 Droplet camera Image 46 Droplet stream calibration 46 E Environmental notices 226 Error messages 218 Ethanol cleaning 188 Ethanol tank 24 refilling 151 Experiment Export 75 Import 76 Searching 77 Sharing 76 F Flip up door 16 Fluidics cart 23 Cart connection cable 24 Ethanol tank 24 Fluidics cart air line connector 24 Sheath tank 24 W
191. hough there is no air trapped in the sheath filter de bubble the sorting chip Click Chip in the De bubble group on the Cytometer tab of the ribbon To release air trapped in the sheath filter manually If the volume of air trapped in the sheath filter is large it may be more efficient to release the trapped air manually before using the de bubble function This procedure is performed with the power supply turned on and SH800 Software up and running 1 In SH800 Software click Settings on the Cytometer tab of the ribbon The Cytometer Settings dialog appears 2 Click Advanced Settings The Advanced Settings dialog appears 3 Click Standby in the Pressure Options tab of the Advanced Settings dialog The sheath fluid flow stops 4 Open the fluidics maintenance door remove the sheath filter from the retaining clamp without disconnecting the connectors and check the fluid level in the filter 5 If the fluid level is low slowly open the air release port on the top of the sheath filter e It is recommended that the drip tray be pulled out partway before proceeding Slowly open the air release port while holding a soft clean cloth ready to wipe away any sheath fluid overflow e Keep the sheath filter clear of all other objects when the air release port is open The trapped air escapes and the fluid level in the sheath filter begins to rise 6 Close the air release port when the fluid level
192. i iat rt The Gate Editor dialog appears Adding Gates 89 se so sishjeuy gs Je deyuo 3 Enter the equation for the Boolean gate on the Boolean Gate tab Enter an equation as follows FITC Gate Editor Gates Click the Equation text box to place the cursor in the box Select a gate in the Gates list and click Select or double click the name of the gate The gate is added to the equation 8 Click AND OR NOT or D as required The logic operator is added to the equation You can also enter the equation directly from the keyboard 4 Repeat steps and to build the equation for the gate Tip The equations for all gates can be viewed on the Details tab Change the name of the Boolean gate as required Click in the Gate Name text box and enter a new name The names of gates must be unique Select a color for the Boolean gate from the Color drop down menu Click Create If the equation specified for the gate is verified as a valid definition for a gate the gate 1s added to the bottom of the gate hierarchy in the Gates and Statistics table Click Close to close the dialog 4 Double click the Boolean gate in the gate hierarchy A default FSC v BSC density plot with linear X axis and Y axis 1s added to the worksheet QQ Adding Gates D Set the axis labels scale types and other properties for the new plot For details see
193. ialog is used to start cleaning of the sorting chip sample line and sample probe using a Sodium Hypochlorite bleach solution For details see Cleaning the Sample Fluidics System using Bleach page 152 Bleach Cleaning Please select cleaning option load the tube on the sample stage and click Start Careful Use 30 ml tube with 25 ml of sodium hypochlorite solution Start Close DI Rinse dialog The DI Rinse dialog is displayed by clicking DI Rinse in the Cleaning group on the Cytometer tab of the ribbon The DI Rinse dialog is used to start flushing of the sorting chip sample line and sample probe using DI water Select the size of conical tube containing DI water to use then click Start to load the tube and begin cleaning DI Rinse Please select rinse option load the tube on the sample stage and click Start Careful Use 30 ml tube with 30 ml of DI water Start Shutdown DI Rinse dialog The Shutdown DI Rinse dialog is displayed by clicking Shutdown Rinse in the Cleaning group on the Cytometer tab of the ribbon The Shutdown DI Rinse dialog is used to clean the sorting chip sample line and sample probe using DI water and then automatically shutting down Select the size of conical tube containing DI water to use then click Start to load the tube and begin cleaning Main Window Shutdown DI Rinse Please select rinse options load the tube on the sample stage and
194. ils about sample tubes sample tube holders and automatic setup bead preparation see Preparing a Sample page 31 2 Press the sample loader door button to open the sample loader door and place the sample tube holder in the sample loader Automatic Calibration Align the D shaped notch on the bottom of the sample tube holder with the slot in the top of the agitation unit when loading the sample tube holder e Always wear laboratory use gloves mask protective goggles and other protective clothing when handling sample fluid to provide protection against biohazards e Also take care when loading the sample tube in the tube holder to avoid sample fluid spills and breakage of the sample tube e Before using a tube check that there is no dust or other foreign matter in the tube e Do not subject the instrument to vibration during operation Shock or vibration may adversely affect alignment and calibration Press the sample loader door button to close the sample loader door Tip The sample loader door will close automatically 1f you leave the door open when you click OK in SH800 Software in the next step to begin calibration Click OK on the Auto Calibration screen Tip If you are planning to analyze samples without sorting them you can select the Analyzer mode Calibrate the chip alignment only checkbox and click OK to bypass sorting calibration This displays the main window in Analyzer mode T
195. ing salt crystal deposits with a cloth may damage the coating Wash the electrode surfaces with water to dissolve salt crystals then wipe clean Electrode surface Clean the deflection plate plastic bolts 8 Spray a lint free laboratory use cloth with ethanol and clean the items in steps 5 to 7 Q Insert the deflection plate in the correct orientation 10 Insert the two plastic bolts and tighten by hand Do not overtighten using a wrench 11 Close the transparent safety cover Cleaning the Waste Catcher The waste catcher in the sorting area should be removed and cleaned when cleaning the sorting area to ensure the area is not contaminated between sorts With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel 2 Open the sorting area door on the front panel 3 Remove the two screws securing the waste catcher and remove the catcher 4 Clean the waste catcher mounting bracket holes and screws using a lint free laboratory use cloth moistened with water 6 Spray a lint free laboratory use cloth with ethanol and clean the items in steps 4 and 5 Wipe any moisture off the screws using a dry lint free laboratory use cloth T Reinstall the waste catcher Cleaning the Waste Catcher puLeuguIeN g seideyuD I 157 eoueuajUley g sajdeyuy 158 e Electrostatic charge may accumulate in the sorting area after cleaning the plastic covers wit
196. ing the probe adapter To reinstall Install the probe adapter Push and hold the sample probe release catch to the right insert the probe straight down and then let go of the release catch Check that the release catch returns all the way to the left 2 Install the PEEK sample line For details about installation see steps 7 to 13 in Changing the PEEK Sample Line PEEK sample line compatible models page 169 Changing the Probe Adapter PEEK sample line compatible models soueuaule g seideyuD I 175 suLeuguIeNw g Ja deuy 176 Changing the Sheath Filter The sheath filter in the fluidics module should be replaced regularly to prevent small particles of dust that can cause fluctuations in the fluid flow and bacteria that can cause contamination of the sample Sheath filter This procedure is performed with the power supply turned on and SH800 Software up and running 1 In SH800 Software click Settings on the Cytometer tab of the ribbon The Cytometer Settings dialog appears Click Advanced Settings The Advanced Settings dialog appears Click Standby in the Pressure Options tab of the Advanced Settings dialog The sheath fluid flow stops Open the fluidics maintenance door and remove the sheath filter from the retaining clamp without disconnecting the connectors Changing the Sheath Filter 5 Disconnect the sheath filter lines from the UPPER a
197. ing Chip The SH800 uses a disposable sorting chip Nozzle size mark SONY Vacuum suction port Sheath fluid inlet port Sample fluid inlet port Optical detection point Nozzle Main Window Sample fluid and sheath fluid enter the chip through inlet ports on the front surface of the chip The sample fluid and sheath fluid are forced into the chip by air pressure from the sample loader and the sheath tank respectively The sample fluid and accompanying sheath fluid flows through the chip where the sample fluid is focused into a stream of single file cells The cells in the sample fluid then pass the optical detection point where they are interrogated by the lasers before being ejected from the nozzle A transducer vibrates the chip at an ultrasonic frequency to break the fluid stream into droplets for sorting A vacuum port provides negative pressure suction to remove bubbles and clogged samples from the fluidics channels in the sorting chip The nozzle size mark indicates the size of the nozzle located at the bottom of the chip The sorting methods that can be used vary depending on the nozzle diameter of the sorting chip and the size of cells For details see Sorting page 53 Do not touch the sorting chip near the optical detection point Fingerprints and smudges near the optical detection point can adversely affect data acquisition results e The menus and buttons displayed vary depending on the user acc
198. ing data i Sample Group 1 aly Tube 2 Next Tube Status Ready Elapsed Time 00 00 00 Total Event 0 _ Start Sample Stop Candition None Click Index Sort Start on the Sort Control pane to start sorting When configured for indexed sorting Index is displayed on the button Sort Control S LA Load Collection Index Sort Start Sorting Method 96 Well Plate Y saa Sort Settings Tip Data is acquired and cells sorted automatically in index sorting B Sample Group 1 a 4 T be 1 Status Recording amp Sorting 00 00 04 Total Event 1 342 Flapsed Time Event Rate Jil cpp Hi les Pause Stop Sample Stop Condition Index Sorting 101 uos 9g Jajdeuy 102 Analyzing Index Sort Data This section describes how to analyze data acquired during index sorting This function allows you to examine the position on plots of cells sorted in a specific well 1 Click Analyze Index Data in the Index Sorting group on the Worksheet Tools tab of the ribbon S rD A CSV Bes Li f nalyze Apply Manua index Data Compensation Compensati e ym mg pa JOT De So ceoH Export io Show CSV File Magnifier a Inra Lrt EAG t Cex A hl u The Index Analysis dialog appears 2 Select a mode in Analysis Mode The wells displayed vary depending on the selected mode Index Analysis Analysis Mode Well Select Mode v s Show All Events
199. installed in the RESERVE slots SH800AC SH800BC SH800DC SH800ZA SH800ZB SH800ZD SH800ZH SH800ZAP SH800ZBP SH800ZDP SH800ZHP 2 On the following models filters not used in the current optical filter pattern should be stored in the RESERVE slots Filter pattern 1 can also be used when the 405 nm laser is turned off to make efficient use of the six fluorescence detection channels SH800CC SH800EC SH800FC SH800ZC SH800ZE SH800ZF SH800ZG SH800ZCP SH800ZEP SH800ZFP SH800ZGP e Filter pattern 1 can also be used when the 405 nm laser is turned off to make efficient use of the six fluorescence detection channels e The shaded portion of the table indicates the filters that are different between optical filter patterns 1 and 2 e Optic filter pattern 2 is used only on the following models equipped with a 405 nm laser SH800CC SH800EC SH800FC SH800ZC SH800ZE SH800ZF SH800ZG SH800ZCP SH800ZEP SH800ZFP SH800ZGP Models other than the above use optic filter pattern 1 only e The longpass optical filters are bigger than the bandpass optical filters to prevent the accidental insertion of optical filters in the wrong slots The LP1 to LP5 longpass optical filters are labeled with the wavelength rating of the optical filter and an LP suffix e g 639LP for 639 nm longpass optical filter The FL1 to FL6 bandpass optical filters are Optical Filter Patterns labeled with the center wavelength of the optical filter
200. ip The PEEK sample line combines the sample line and sample probe in a single component The sample line and probe are maintenance items Name and Function of Parts 17 roneo wi WN M Q Je deyD 18 Make sure the sample line is fully inserted into the pinch valve Failure to do so may result in fluid leakage Touching the sample line will directly affect the chip alignment If you touch the sample line after the chip alignment has been adjusted the chip must be realigned using the chip alignment function Always leave the sample line connected when the unit is not in use for protection against dust For details see Changing the PEEK Sample Line PEEK sample line compatible models page 169 or Changing the Sample Line and Probe Non PEEK sample line compatible models page 172 Sample probe release catch Used when removing the sample probe or probe adapter For details see Changing the Sample Line and Probe Non PEEK sample line compatible models page 172 or Changing the Probe Adapter PEEK sample line compatible models page 175 1 Sorting area SH800 and SH800Z models Name and Function of Parts Detection module Comprises an array of dichroic beam splitter longpass filters LPF to separate the collected fluorescent light into separate channels and bandpass optical filters BPF to filter the light entering each detector There are eight detectors forward sca
201. ip in the correct orientation and try loading again If the problem persists contact your Sony distributor for service Check the waste fluid line connection to the waste catcher If the problem persists contact your Sony distributor for service Inspect the drip tray and check for any fluid leakages If the problem persists contact your Sony representative for service Check that the connectors for all fluidics lines in the fluidics cart and main unit are connected securely and that the lid of the sheath tank is closed tightly Check that the black rubber seal is attached to the lid of the sheath tank Check that the sorting chip was not inserted back to front Check that the appropriate pressure is supplied to the instrument Supply pressure 500 kPa to 650 kPa 73 psi to 94 psi If the problem persists contact your Sony distributor for service Insert the chip in the correct orientation and try loading again If the problem persists contact your Sony distributor for service Error Messages sno ueLjj v s y g xlpueddy 221 sno ueLjj s g xipueddy 222 GUI Error Message Recommended Solution Failed to release the chip Shut down the cytometer and turn off the compressed air l supply alee Cip Eror rallen io Mote RIR Pull the sample line connector out slightly from the chip Unload Chip Error Failed to release the chip loader and turn the Emergency thumbwheel to eject the chip The model numb
202. itation laser module uses Automatic Power Control APC to provide optical measurement accuracy and reproducibility Intensity Stokes shift Emission spectra Excitation spectra Beam combiner Wavelength Optical fiber Objective lens module Optical System s jd uld BuryessdoQ y xipueddy 197 sojdiouudg BuryesodoQ y xipueddy 198 Light collection Laser module Detection module Fluorescent light BSC light FSC light Optical fiber Optical fibers Delivery Objective FSC unit optics Numerical Aperture NA 0 85 Sorting chip The objective lens module focuses the beam from the laser module onto the optical detection point in the sorting chip The laser light source from the excitation laser module is focused on the optical detection point in the sorting chip Detection The detection module takes the light collected from the objective lens module and separates it into eight channels of light comprising forward scatter light FSC back scatter light BSC and six channels of fluorescent color light FL1 to FL6 The output from each channel is received by a detector that converts the light energy into an electrical signal for processing by the acquisition module Objective lens Dichroic mirror module Optical fiber ae ay FSC The detection module is comprised by an array of dichroic longpass optical filters LPF and bandpass
203. ition Stop condition Selects the number of events before recording stops automatically User Defined Color tab Specifies custom colors for gates User Preference sant Define user defined color for gate Reset Cytometer User Defined Color Experiment Worksheet BEES Compensation FCS File tab Specifies default settings related to FCS files User Preference Sort Set default settings for FCS file Reset Cytometer Export FCS Files Experiment Set default export folder of FCS files Export Folder Worksheet Compensation User Defined Color Auto FCS Exporting Function FCS File FCS file will be automatically generated when acquisition stops A To maximize the security of your data please ensure that auto exported data are stored in a defined folder Enable Auto FCS Exporting Export FCS Files Export Folder Specifies the destination folder used when exporting FCS files Auto FCS Exporting Function Enable Auto FCS Exporting Enables the automatic exporting of experiment data to FCS files Note that data security may be reduced if automatic export is enabled Export Folder Specifies the destination folder used when exporting FCS files File Name Format Setting Specifies the file naming format You can check the file name in Preview e Include Experiment Name e Include Sample Group Name FCS Version Selects the FCS file version e 3 0 e 3 1 Convert BSC
204. ition status For details about the displayed items see Acquisition Tab control pane page 132 Acquiring Data B Sample Group 1 o Tube 1 Status Pause Elapsed Time Total Event Event Rate 0 eps a Restart Start Stop Record le Stop Condition None v Start Starts resumes data acquisition It does not start data recording Pause Pauses data acquisition The elapsed time continues counting while acquisition is paused Stop Stops data acquisition manually Record Records the acquired data REC stop Stops data recording manually Restart Resets the total events and elapsed time counters to 0 and restarts data acquisition Tip If an automatic stop condition has been specified data acquisition and or recording stops automatically when the corresponding stop condition has been satisfied Customizing the Worksheet You can change the type of plots modify gates on plots and customize the content in the Gates and Statistics table displayed on the worksheet during data acquisition You customize the items on the worksheet using the tabs on the ribbon Example Worksheet Tools tab an 2 y Syl Experiment Cytometer ompensation Tube 1 Data Source 1 All Events i jen We e E3 Acquisition 5000 GX QR 9 ons wW w csv jew New Dot New Remove Duplicate Analysis 100 000 v Remove Edit Shi d Ex Statistic nsity Plot Histogram Plot Piot Gate Gi Ta
205. keywords as required Required FCS text segment keywords are exported automatically The following optional FCS text segment keywords can be specified e INST Institution at which data was acquired EXP Name of investigator initiating the experiment e OP Name of instrument operator e PROJ Name of the experiment project e SMNO Specimen e g tube label e SRC Source of the specimen patient name cell type CELLS Description of objects measured e COM Comment Output Folder Specifies the export destination folder Click Browse to specify the destination folder The files are named in lt tube_name gt fcs format O Progress Displays the export progress Export Starts the export Close Closes the dialog Cytometer Tab ribbon The Cytometer tab of the ribbon has the following buttons Load Unload group Contains tool buttons for loading and unloading sample tubes and the collection stage Load Collection Unload Collection Loads unloads the collection stage in the sorting area Load Sample Unload Sample Loads unloads the sample tube in the sample loader Main Window uondos q mopuiM Z JeldeyD I 117 uoldiuoseg MOpUIN Z Je deuyD 118 Light group Contains tool buttons for turning the collection stage and sample loader lights on off 5 5 F E ai P 1 T Collection Sample Collection Turns the collection stage light on off Sam
206. l analysis and sorting The operator uses the software to create experiments for measuring data and to create plots and define statistics for analyzing data The export functions are fully compliant with Flow Cytometry Standard version 3 0 or 3 1 The software provides quick access to all functions from the main window The SH800 has a compact footprint size The benchtop unit has external dimensions W x D x H of 550 x 550 x 720 mm 21 3 4 x 21 3 4 x 28 3 8 in and weighs 100 kg 220 Ib Exposing the main unit or the fluidics lines between the unit and the fluidics cart to drafts from a room air conditioner may cause droplet formation to become unstable Tip This document describes the basics of flow cytometry as it pertains to understanding how the SH800 cytometer operates It is not intended as a reference on the subject of flow cytometry There are many reference sources available both in print and online that you should consult for flow cytometry specifics such as sample preparation dye selection analysis techniques results publication methods and other topics Main Features 13 Gaa WN M Q Je deyD Cell Sorter Block Diagram 14 Fluidics tanks Sorting chip Sample tube SOK SAE BAY 3 NZ 152 57 Siess Waste Ethanol Sheath DI water tank tank tank tank Fluid supply Air supply Waste collection Sample fluid Sheath fluid Sample loader Ch ader Fluidics system aad E ip lo H
207. laboratory use gloves mask protective goggles and other SH800ZP only tective clothing a Pree One ane Attach the supplied splash guard when sorting into multi Note well plates to prevent splashing of sample fluid Before using a tube check that there is no dust or other foreign matter in the tube Loading 8 well Strips SH800 and 4 SH800Z Observe the following precautions when loading 8 well strips Insert the leading edge of the 8 well strip into the holder at an angle and push the plate in all the way Splash guard 1 Hold the splash guard in the orientation shown in the diagram and insert the protrusion on the splash guard into the notch on the waste catcher Waste catcher 2 Orient the holder with the FRONT arrow towards you and place the holder on the collection stage Instrument side Align protrusion and notch Si Front side Preparing Collection Tubes 35 uoneiedaig Z19 deyo HIN uoneledaig zguiajdeyy 36 Remove the splash guard when unloading multi well plates after sorting is finished Preparing Collection Tubes Loading a Multi well Plate SH800ZP only Place the multi well plate into the plate holder with the chamfered corners toward the rear of the instrument Rear of instrument Multi well plate holder A1 e Always use a 384 well plate holder when using 384 well plates Do no
208. le g Ja deuy 190 Connect the sheath filter bypass line yellow and DI 12 Remove the sheath filter bypass line yellow and DI water filter bypass line green to the corresponding water filter line green and reattach the sheath filter UPPER and LOWER connectors and DI water filter Yellow Green The DI water filter goes on the right and the sheath filter on the left Check that the filters are not connected incorrectly Sheath filter DI water filter 8 Hold the QR code on the packaging for the cleaning chip in front of the built in camera on the PC 13 Click Next The currently loaded sorting chip if one is present is automatically ejected The SH800 hardware and software shut down automatically O Insert the cleaning chip in the insertion slot on the The cleaning chip remains loaded in the chip loader top of the chip loader then click Next after shutdown 1 0 Enter the ethanol cleaning time then click Start The recommended ethanol cleaning time is 12 minutes A progress bar is displayed during cleaning The sheath fluid line is cleaned with ethanol for the specified duration When the cleaning time has elapsed the sheath fluid line is flushed with DI water to remove all traces of ethanol 11 When cleaning is completed click Next Cleaning the Internal Sheath Line and DI Water Line using Ethanol Operating Principles This chapter describes some of the basic concepts of flow cytomet
209. le please stop acquisition problem occurs stop data acquisition The droplet condition has changed since the last Sort The formation of droplets has changed since auto calibration Calibration The Sort Calibration should be performed again was last executed Run Sort Calibration again to ensure for stable sorting stable sorting conditions Use only within the specified temperature range Do not use under conditions where the temperature is subject to rapid changes Failed to initialize the sheath pressure regulator Sheath Shut down the main unit and wait a while before trying again pressure is too high If the problem persists contact your Sony distributor for Please restart the cytometer and software service If the error reoccurs please contact your service representative Failed to initialize the sample pressure regulator Sample pressure is too high Please restart the cytometer and software If the error reoccurs please contact your service representative High sheath pressure is detected while initializing the An unusual pressure value was detected You can continue regulator with operations but you should contact your Sony distributor Please contact your service representative for service High sample pressure is detected while initializing the regulator Please contact your service representative Low sheath pressure is detected while initializing the regulator Please contact your service representative Lo
210. lick Manual Compensation again to deactivate manual compensation mode You can also deactivate manual compensation mode by selecting another worksheet to display Adjusting Fluorescence Compensation 81 sluewuedxy Buunbyuoy p Jaldeyy iii s yu wn dx Buunbyuog p Je deyD e Adjusting the position of events shown on a plot also adjusts the position of all events on other plots Accordingly you should add plots for all fluorescence parameters to the worksheet before adjusting compensation manually in order to monitor the affect of adjusting the compensation on each plot e The actual compensation values in the spillover matrix can be monitored simultaneously in the Compensation Settings dialog It is recommended that you save the compensation settings from the Compensation Settings dialog before adjusting compensation manually You can also click Reset in the Compensation Settings dialog to reset the compensation settings To edit the spillover matrix directly You can adjust the fluorescence compensation manually in the Compensation Settings dialog You can adjust the values using visual feedback alone or enter spillover matrix values directly to reproduce the compensation settings of another experiment Editing the spillover matrix directly to adjust compensation is inherently difficult Exercise care when editing the spillover matrix directly 1 Display the worksheet for the tube whose compensation you want
211. ll affected surfaces The system must be restarted after a certain time has elapsed after the alert is raised DI water filter Filters the DI water flowing from the DI water tank to the sample probe and other components The DI water filter is a maintenance item DI water filter Name and Function of Parts 21 roneo va WIN M IA AO Je deyD 22 Rear panel Main power switch 1 Fluidics cart connector block Heat exhaust vent AMS evacuator adapter connection point AC power connector AC IN PC connector cable connection Main power switch MAIN POWER Turns the power supply for the SH800 ON up and OFF down The breaker automatically disconnects the power supply if an electrical fault is detected AC power connector AC IN Connects to a standard AC power supply 100 V to 240 V AC 50 60 Hz Use only the supplied power cord to connect the SH800 main unit to a power supply e Use only within the specified supply voltage range e The power cord must be connected to a power outlet with an Earth connection PC connection cable connector PC CONNECTION Connects directly to the host computer using the supplied PC connection cable Do not disconnect the PC connection cable during operation Data acquisition cannot be performed if the cable is disconnected Do not connect any device other than the host computer using the PC connection cable Device operation is
212. lot Quadrant Creates a quadrant gate on a dot plot or density plot Linear Creates a linear gate on a histogram plot Plot Type gt Density Changes the type of the selected plot to a density plot Dot Plot Changes the type of the selected plot to a dot plot Histogram Changes the type of the selected plot to a histogram plot Zoom Zooms in on the area selected by dragging the mouse on a plot Open Gates Editor Displays the Gate Editor dialog page 127 to edit gates Open Statistics Editor Displays the Statistics Editor dialog page 92 to configure the statistics displayed in the Gates and Statistics table Copy Picture Copies the selected plot to the clipboard for pasting in other software for example to produce reports Properties Displays the Property Window dialog page 130 Gates The following menu commands are displayed when right clicking a gate Create Density Plot Adds a density plot for the events within the selected gate Create Dot Plot Adds a dot plot for the events within the selected gate Create Histogram Plot Adds a histogram plot for the events within the selected gate Move Color Order to Front Moves the selected gate to the front Move Color Order to Back Moves the selected gate to the back Convert to gt Rectangle Converts the selected gate to a rectangle gate Ellipse Converts the selected gate to an ellipse gate Polygon Con
213. loth ready to wipe away any DI water overflow e Keep the DI water filter clear of all other objects when the air release port is open The trapped air escapes and the fluid level in the DI water filter begins to rise 14 Close the air release port when the fluid level nears the top of the filter Make sure that the air release port is tightened and that there is no leakage of fluid If the air release port is overtightened the filter may become damaged Do not apply excess force when tightening 15 Click Stop 1 6 Place the DI water filter in the retaining clamp and close the fluidics maintenance door 17 When finished click Close Changing the Sintered Sheath Line Filter A sintered sheath line filter is placed in the sheath line to filter residue or small particles from the sheath fluid supply The filter should be replaced every 12 months Sintered sheath filter 1 With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel of the main unit Make sure the main unit is completely shut down before proceeding 2 Turn off the compressed air supply 3 Hold the sintered sheath line filter with one hand push and hold in the quick release collet with the other hand and pull out the sheath fluid line blue from the side furthest from the sheath tank Exert gentle force if the tube feels difficult to pull out from the connector Changing the Sintered
214. lowing buttons Refer to the Worksheet Tools tab for a description of the Gate group buttons page 126 Label group Contains tool buttons for specifying gate labels Font Size 10 pt T Statistic Parent Label Font Size Selects the font size of text labels Statistic Selects the statistic that is displayed beside gates on plots Type Convert group Contains tool buttons for converting the selected gate to a different gate type _ To Rectangle To Polygon Style group Contains tool buttons for changing the display style of gates EEHEEHE h 1 Visible Shows hides the selected gate Color palette Changes the color of the selected gate Edit Color Allows you to edit the color in the lower color palette user defined colors to any color Click a color in the color palette click Edit Color then select a new color and click Apply yy Ee F 5 E R 2 ui _ Edit Width Move to to Remov Color 1 Front Back Gate inde FFADAODAD Width Selects the line width of the selected gate Color Order group Contains tool buttons for changing the display order of gates uf iy Mowe to Front Move to Front Moves the selected gate to the front Move to Back Moves the selected gate to the back Tools group Contains a tool button for copying gates Dy Copy Took Copy Copies the selected gate Gates and Statistics Table The Gat
215. lt worksheet settings Favorite Worksheet Settings Modified Displays the date and time that the favorite worksheet settings were saved Preview Displays a preview of the worksheet with the saved favorite worksheet settings Close Closes the dialog Stop amp Sort Settings dialog The Stop amp Sort Settings dialog is displayed by double clicking Stop amp Sort Settings for a sample tube in the Experiment Explorer The stop and sort settings can be viewed in the Stop amp Sort Settings dialog Example 2 way tube sorting Tube 1 Stop amp Sort Settings Sample Stop Condition None Recording Settings Crann ji Collection Tube Sort Gate Sort Mode Cell Size Stop Count Sample Stop Condition Displays the data acquisition stop condition Recording Settings Stop Condition Displays the recording stop condition Main Window uondos q mopuiM Z JeldeyyD I 135 uoldiuoseg MOpUIN Z Je deuyD 136 Sort Settings Sorting Method Displays the sorting method for the sample tube You can change the order items are displayed by dragging and dropping the title cells Close Closes the dialog Sorting Control Pane The sorting control pane is used to configure and control cell sorting For details see Sorting page 53 The items displayed in the sorting control pane vary depending on the selected sorting method This section describes the displayed items using two
216. ly tighten the cap and stow the tray Make sure that the waste fluid line is connected securely to the connector on the cap of the waste tank and that the cap is securely fastened on the waste tank to prevent leakages Always place the tank in the center of the tray area The fluid level detector may not operate correctly if the tank is placed off center Do not autoclave fluid tanks The shape of the fluid tanks will deform if placed in an autoclave If the air filter attached to the waste tank becomes blocked air cannot escape the waste tank as it fills with waste fluid which may cause incorrect operation The air filter may become blocked if it gets wet Do not tilt or otherwise jostle the waste tank if it is close to full Refilling the DI Water Tank The DI water tank should be refilled daily before starting the unit The tank should also be refilled as required if the level becomes too low during operation and before cleaning with ethanol Tip The DI water tank can be refilled at any time except during auto calibration measurement and cleaning Precautions when handling DI water Refill the DI water tank with fresh DI water every time when starting up replacing any unused DI water in the tank Regularly clean the DI water tank with ethanol to suppress the growth of bacteria page 166 Use ultra pure water or distilled water with extremely little or no bacteria or biological content to further suppress the g
217. manually 3 Select the gate whose population you want to sort into the well in Sort Gate You can change the color of the gate in Color Select a sorting mode in Sort Mode The default is Single Cell Single Cell mode is recommended for reliable sorting Select the size of the cells to be sorted in Cell Size As a rough guide select Regular Cell for cells up to 15 um in size and Large Cell for larger cells When you select Large Cell a charge setting suited for sorting larger cells is used However this function does not guarantee complete accuracy in sorting large cells Enter the number of events to sort in Stop Count If a value exceeding the values below is entered in Stop Count a message appears warning you of the risk that sample fluid may overflow the well operation does not stop Sorting chip 8 well strips Slides 100 um nozzle 50 000 events 300 events Enter the timeout time in Timeout in units of seconds Click Add The settings are added to Sort ID List Repeat steps to to set parameters for any other wells if necessary You can modify the parameters for a well by clicking the corresponding cells in Sort ID List When finished click Close to close the dialog Click Start or Resume in the data acquisition control pane to begin data acquisition Status Ready 00 00 00 Total Event 0 Flapsed Time Event Rate 0 eps Sa
218. matic zoom tracking function Changes the shape and size of gates on plots to track the zoom size of plots and change of axis type Default Scatter Plot Plot Type Selects the default type of plot added to a new worksheet e Density e Dot Plot Axis Type Selects the default scale type of an axis e Linear e Log logarithmic e Biexponential Gate type Selects the default gate type e Ellipse e Polygon e Rectangle Index Analysis Plot Size Specifies the size that events are displayed on plots for emphasis when using the index sorting function You can check the size in Preview Alignment Auto Arrange Selects whether to automatically align the plots and Gates and Statistics table to the grid of the worksheet e On e Off Snap to Grid Selects whether to align the plots and Gates and Statistics table to the grid when moving or adding them on the worksheet e On e Off Show Grid Selects whether to display the grid on the worksheet The size of the grid is set in Grid Size e On e Off View Expand Sets the zoom factor of the worksheet display Compensation tab Defines default settings related to fluorescence compensation User Preference Sort Set default setting for compensation Reset Cytometer Recording Stop Condition Experiment Set default recording stop condition of compensation tube Worksheet User Defined Color FCS File Recording Stop Cond
219. ment adjusting sort parameters and other instrument functions Droplet Displays a detailed image from the droplet camera Help group Contains a tool button for displaying the optical filter pattern aa Filter Settings Help Filter Settings Displays the configured optical filter pattern This cannot be modified Shutdown group Contains tool buttons used when shutting down the SH800 lt x si lal i d d Hardware Software and Only Hardware Fthana Cleaning Hardware Only Displays the Shutdown Wizard screen for cleaning the SH800 and then shutting down the instrument only For details see Shutdown page 65 Software and Hardware Displays the Shutdown Wizard screen for cleaning the SH800 shutting down the instrument and SH800 Software For details see Shutdown page 65 Ethanol Cleaning Displays the Ethanol Cleaning wizard for cleaning the internal sheath fluid line and DI water line with ethanol The SH800 instrument and SH800 Software automatically shut down after cleaning is finished For details see Cleaning the Internal Sheath Line and DI Water Line using Ethanol page 188 Main Window uondos q mopuiM Z JeIdeyD I 119 uolduoseg mopuiM Z Je deuyD 120 Bleach Cleaning dialog The Bleach Cleaning dialog is displayed by clicking Bleach Cleaning in the Cleaning group on the Cytometer tab of the ribbon The Bleach Cleaning d
220. move the support guide Please reload the tube If the error reoccurs please contact your service representative Load Sample tube Error Failed to move the sample stage Please reload the tube If the error reoccurs please contact your service representative Sheath Fluid Tank is empty Refill the sheath fluid tank following the instructions in the operator s guide Waste Fluid Tank is full Empty the waste tank following the instructions in the operator s guide Ethanol Fluid Tank is empty Refill the ethanol fluid tank following the instructions in the operator s guide Compressed air is not supplied Confirm if the compressed air hose is connected or the sheath tank is set up properly Fluidics cart is not connected Connect the fluidics cart correctly following the instructions in the operator s guide The ambient temperature exceeds the design operating range Shut down the cytometer The temperature of the sample area is too high The temperature of the collection area is too high Sheath leakage is suspected Check that the fluidics cart is solidly connected and if there is sheath leakage inside the cytometer The sheath tank on the fluidics cart is not detected Check that the sheath tank is attached The waste tank on the fluidics cart is not detected Check that the waste tank is attached The temperature control of the sample area failed The temperature control of the collection area failed
221. mum measured values Auto Adjust X Axis Auto Adjust Y Axis Automatically adjusts the range of the X axis or Y axis respectively of the selected plot to the minimum and maximum measured values Zoom Zooms in on the area selected by dragging the mouse on a plot Default Adjust Restores the default X axis and Y axis ranges Click the down arrow to set the number of decades displayed on log scales ai Displays the Property Window dialog page 130 for specifying axis types and scale ranges Gate group Contains tool buttons for drawing gates on plots and editing gate properties HOA H GR Rectangle Ellipse Polygon Quadrant Linear Remove Edit Gate Gate For details see Adding Gates page 88 and Editing Gates page 91 Rectangle Creates a rectangular gate on a dot plot or density plot Ellipse Creates an elliptical gate on a dot plot or density plot Polygon Creates a polygonal gate on a dot plot or density plot Quadrant Creates a quadrant gate on a dot plot or density plot Linear Creates a linear gate on a histogram plot Remove Gate Deletes the selected gate Deleting a gate will also delete all gates and plots that are derived from the selected gate Edit Gate Displays the Gate Editor dialog page 127 for editing gates Plot View group Contains tool buttons for changing the display of plots woo re 5 Change Palette Si x20 Plat Wiew
222. n a separate window for monitoring progress Elapsed Time Displays the cumulative elapsed time since the start of sorting Remaining Time Displays the estimated remaining sorting time Sort Count Displays the number of events sorted Sort Rate Displays the rate at which events are detected during sorting in units of events per second eps Sort Efficiency Displays the number of sorts attempted as a percentage of targeted cells in the sample Abort Count Displays the number of events that do not match the sort criteria and which are aborted during sorting Abort Rate Displays the rate at which events are aborted during sorting in units of events per second eps For multi well plate sorting The following items are displayed when a multi well plate is selected in Sorting Method Example When 96 Well Plate is selected 2 The Droplet and Hardware Status panes are the same as for two way tube sorting Sort Control pane Configures and controls droplet sorting Sort Start Sort Stop Starts stops sorting Load Collection Unload Collection Loads unloads the collection stage Sorting Method Selects the sorting method Set the sorting method to match the collection devices loaded on the collection stage The sorting parameters vary depending on the selected sorting method Main Window uondos q mopuiM Z JeIdeyD I 137 uoldiuoseg MOpUIN Z Je deuyD 138
223. n as the default click Save Custom Position Tip Clicking displays the User Preference dialog page 105 for setting user default preferences 4 When finished click Close to close the dialog Select the type of test sorting in Sort Test gt Sort Test Type e Selected Target Well Droplets fall in the well position selected in Select Target Well Select this option to test sort into a single well at a time When selected select the well into which droplets should fall in Select Target Well e Four Corners and Center Well Droplets fall into the four corner wells and the center well e One fourth of All Well Droplets fall into 1 4 of all wells e All Well Droplets fall into all wells 2 Click Start in Sort Test Change the number of events to be sorted into each well in Count as required Droplets start to fall in the position selected in step 3 Click Unload 4 Visually check the position of the droplets on the multi well plate cover Wipe off droplets after confirming their position Adjust the sort position of droplets using the arrow buttons in Droplet Position For example if the position for well Al was 1mm too far left and 1 5mm too far up 1 Click well Al Adjusting the Sort Position SH800ZP only Index Sorting You can associate the cells sorted in each well with the data points for those cells on plots Using the index sorting fu
224. n droplet and adjacent and subsequentdroplets are empty Not sorted Target cells are in adjacent or subsequent droplets COWL Sorting Mode 95 uos 9g Jajdeuy Normal Mode Normal mode is the default sorting mode 100 um sorting chip A droplet is sorted if it contains one or more targeted events of a single type One droplet is sorted for each event matching the sorting criteria 3 Not sorted Conflicting cell is in ale eae ac droplet 130 um sorting chip A droplet is sorted if it contains one or more targeted events of a single type In addition if the targeted event is near the edge of the droplet the nearest adjacent droplet is also sorted if that droplet is empty or non conflicting One or two droplets are sorted for each matching sorting criteria Sorted Target cell is outside center of main droplet and nearest adjacent droplet is empty Not sorted Conflicting cell is in droplet regardless of target cell in nearest edge of main droplet Sorted Target cell is within center of droplet Sorting Mode Semi Purity Mode In Semi Purity mode the purity achieved is higher than in Normal mode 100 pm sorting mode A droplet is sorted if it contains one or more targeted events of a single type and the nearest edge region of both of the adjacent droplets is empty or non conflicting One droplet is sorted for each event matching the sorting criteria iG ie N Sorted
225. naked flame embers or other material that can emit sparks near the main unit or the fluidics cart Ethanol vapor is highly flammable at normal room temperature e Do not pour any fluid other than the specified fluid into the fluidics system tanks e Make sure that the cap on the ethanol tank is correctly and securely fastened at all times e Make sure that the ethanol line is securely connected to the connectors on the rear panel of the main unit rear panel of the fluidics cart and the ethanol tank itself e Make sure that the cap on the waste tank is correctly and securely fastened at all times e Make sure that the waste fluid line is securely connected to the connectors on the rear panel of the main unit rear panel of the fluidics cart and the waste tank itself The SH800 is compatible with standard disinfecting agents ethanol 70 for use in flow cytometers 1 Click Maintenance on the bottom right of the SH800 Software login screen Cell Sorter SH800 The Maintenance wizard appears 2 Select Ethanol cleaning then click Start Maintenance Please select maintenance mode Ethanol cleaning Internal sheath line and DI water line are cleaned with ethanol WasteA maintenance D It is recommended that if you have not used for a long time to perform WasteA maintenance Sample line exchange Exchange sample line and probe for new one The Ethanol Cleaning wizard appears 3 Click Start
226. nce compensation e Data source indication e Sample empty detection function e Maintenance mode Functions Added in Version 1 5 The following functions have been added in SH800 Software version 1 5 Multi well plate support SH800 96 well plate model Sample fluid can be sorted into 6 12 24 48 and 96 well plates For details see Preparing Collection Tubes page 33 and Sorting page 53 Index sorting An index sorting function has been added that links the data for sorted events to the individual well in which the events were sorted For details see Index Sorting page 101 Specifying a gate as calculation target for fluorescence compensation This function allows you to specify the event population for a gate as a negative or a positive control used for calculating fluorescence compensation For details see Using a Target Gate in the Gate Hierarchy page 80 Data source indication A data source component has been added to the Experiment Explorer that indicates the type of sorting collection device for saved data For details see Data sources page 140 Sample empty detection function This function detects when the level in the sample tube is too low and stops data acquisition automatically For details about each item see Sample Empty Detection page 123 in the Advanced Settings dialog Maintenance mode This function allows an operator to run eth
227. nconsciousness Never place a naked flame embers or other material that can emit sparks near the main unit or the fluidics cart Ethanol vapor is highly flammable at normal room temperature Never pour ethanol into a tank other than the ethanol tank and conversely never pour other liquids into the ethanol tank Make sure that the cap on the ethanol tank is correctly and securely fastened at all times Make sure that the ethanol line is securely connected to the connectors on the rear panel of the main unit rear panel of the fluidics cart and the ethanol tank itself Make sure that the lid on the waste tank is correctly and securely fastened at all times Make sure that the waste fluid line is securely connected to the connectors on the rear panel of the main unit rear panel of the fluidics cart and the waste tank itself The SH800 is compatible with standard disinfecting agents ethanol 70 used for flow cytometers 1 Replace and empty the waste tank For details see Emptying Changing the Waste Tank page 148 Launch SH800 Software On the Auto Calibration screen click Skip Auto Calibration to bypass automatic calibration Click Settings on the Cytometer tab of the ribbon The Cytometer Settings dialog appears 4 On the Control tab click Advanced Settings Cytometer Settings xX Chip Information Cytometer Information Maintenance Laser Threshold I 405nm On Tumo Ch
228. nction allows you to select a specific well and view the data for cells collected in that well This section describes sorting using a 96 well plate but the procedure is identical for sorting onto 8 well strips or 384 well plates Tip Index sorting is not available for 2 way tube sorting Configuring Index Sorting Select a sorting method in Sorting Method in the Sort Control pane and click Sort Settings nrd Sort Control amp vw Load Collection Sortmg Method 96 Well Plate The Sort Settings dialog appears 2 Place a check mark in Add index sort information on the Plate Sort Settings tab Sort Settings 96 Well Plate Plate Sort Settings Plate Adjustment Index Sort Sort Layout Settings Sort Layout Settings Column to Row A1 gt H1 E Row to Column A1 gt A12 Sorting Target Well lI Sort ID Sort ID 3 000000000000 Sitti A 000000000000 e a 1000000000008 oa _ OOOO 0O00009 Sort Mode Single Cell 1900000000000 as EEA 00000000000 eas OOODO0OOOOO8 H 000000 Oo Oo ee Timeout 0 Seconds 1 23 amp S 7 8 9 10 1E 2 Add Sort ID List Sort ID Sort Gate Color Sort Mode Cell Size Stop Count Stop Count Sort ID 1 A HM Single cell Regular Cell 100 Sort ID 2 8 BE Single cell Regular Cell 100 Sort g R 1 3 When finished configuring other items click Close to close the dialog A Click Start on the data acquisition control pane to start acquir
229. nd LOWER connectors Press the metal release catch and withdraw each connector The sheath filter connectors have stop valves that prevent leakage of fluid when disconnected 6 Discard the used sheath filter Discard the sheath filter responsibly in accordance with local and federal ordinances and regulations 9 Hold a new sheath filter with the arrow on the side of the filter facing up Connect the two fluidics lines from the sheath filter to the corresponding connectors Connect the line from the top of the filter to the UPPER connector and the line from the bottom to the LOWER connector Insert each connector and push it until it locks into place A click sound occurs when the connectors lock into place Place the sheath filter in the retaining clamp 10 In SH800 Software click Sheath Filter in the De bubble group on the Cytometer tab of the ribbon The Sheath Filter De bubble dialog appears 11 Click Start When the de bubble function is completed click Close Repeat two times to remove residual air from the sheath filter The sheath filter de bubble process takes a short time to finish Run the sheath filter de bubble function at least three times to make sure that all air and small particles have been removed from the filter and sheath line Failing to do so may cause any solid matter not removed from accumulating and clogging the sorting chip 12 Perform steps 4 to 8
230. nd closing the door while the power is turned off make sure the compressed air supply is turned off Failure to do so may result in damage or injuries O Power button POWER STANDBY Switches the SH800 main unit power supply between ON and OFF When power is ON press and hold for about two seconds to switch Power OFF Display Mode button Switches the LCD monitor display on off e Do not place any objects in front of the doors LCD monitor Displays instrument status information For details see LCD Monitor page 28 e Be aware of the door edges when the flip up door sorting area door and fluidics maintenance door are open Internal View Front The following illustration depicts the SH800ZP Chip loader ho Pinch valve 1 Sorting area Chip loader Loads and ejects the sorting chip The chip is inserted in a top loading slot Sorting chip l EMERGENCY LOAD UNLOAD Chip insertion slot Sorting chip manual load unload thumbwheel Detection module Sample probe release catch 2 Sample loader area The chip unloading thumbwheel is used to eject the sorting chip if the chip will not unload automatically For details see Ejecting the Sorting Chip Manually page 185 Pinch valve Controls the flow of sample fluid between the sample probe and the sorting chip Sample line Transports the sample fluid from the sample loader tube to the sorting ch
231. nd scratches for accurate data acquisition and cell sorting operation For details about handling optical filters see Cleaning and Handling of Optical Filters page 186 RESERVE slots e On the following models dummy filters are installed in the RESERVE slots SH800AC SH800BC SH800DC SH800ZA SH800ZB SH800ZD SH800ZH SH800ZAP SH800ZBP SH800ZDP SH800ZHP e On the following models filters not used in the current optical filter pattern should be stored in the RESERVE slots Filter pattern 1 can also be used when the 405 nm laser is turned off to make efficient use of the six fluorescence detection channels SH800CC SH800EC SH800FC SH800ZC SH800ZE SH800ZF SH800ZG SH800ZCP SH800ZEP SH800ZFP SH800ZGP For details about filter pattern I and 2 see Optical Filter Patterns page 202 B Click Next The Fluidics Check screen appears Initial Instrument Setup vV 1 Chip Detection Please check that sheath is dripping from the tip of sample probe If not click Sample line cleaning to start Bleach Cleaning and DI Rinse to clean sample line vV 2 Chip Information vV 3 Chip Loading V 4 Laser Setting V 5 Filter Setting Fluidics check is in progress 6 Fluidics Check When fluidics check starts sheath fluid droplets appear from the tip of the sample probe If droplets do not appear the sample line or the sample probe may be clogged Click Sample line cleaning on the bottom right of t
232. nd width of the pulse Hence the optimum window extension will vary from pulse to pulse within the same sample and from experiment to experiment If the window extensions are too narrow the measurement for the area pulse parameter will be smaller than its real value If the window extensions are too wide then the window extension may begin to overlap with other pulses degrading the calculation When a cell is detected during sorting it is generally not possible to know with great accuracy if the signal represents the passage of a large single cell small multiple cells adhering to one another or other non cellular material Accordingly it is more common to refer to each signal detection not as a cell but as an event Fluorescence Compensation When detecting the emitted fluorescent light for a fluorochrome components of the emitted light can appear in one or more of the fluorescent light detectors FL1 to FL6 due to the wide emission spectrum of each fluorochrome When detecting the emitted light for more than one fluorochrome components of the spectrum of all fluorochromes can appear in multiple detectors a Optical System sojdiouud BuryessdoQ y xipueddy 199 sgjdiouudg Buryeiodo y xipueddy 200 phenomenon called spillover The problem then becomes one of working out the proportion of the light detected in each detector due to the individual fluorochromes For example in the following diagram the output of d
233. ne ae OEN 69 Chapter 4 Configuring Experiments Creating a Customized Experiment ccssesesseeeeeees 70 Configuring an Experiment cccccssseceeseeessesseseeseesesens 73 Editing an Experiment ccssccsssscsessccsesseesssesseeasenaseeeaes 74 Editing a Sample Group sssrinin ai 74 Edine a Sample DUDE armeria asetnohs dalatanie he adaels 74 Changing Component Settings 00 0 ceeeecssecceceeeeeeeseseeeeeeees 75 Exporting Importing an Experiment ccsseeseeseeeees 75 Exporting an ExXperinicmt nisssccvat eons inten hearse 75 limportins an EX periment erisso urna r e 76 Sharing Experiments ccccccsscessseeseeseccesseeeesseeeeseeseasesees 76 Searching Experiments ccccssseecsesseeseenseesensseeseesseesees 77 Saving Tube Data aS FCS Files ccssesseeesesseeeeeseeeeees 77 Saving an Experiment as a Template cssseeeeeeees 78 Configuring Detector Settings ccccssseessssseeeseeseeseeees 79 Adjusting Fluorescence Compensation c0000000 80 Using a Target Gate in the Gate Hierarchy ceeeee 80 Adjusting Fluorescence Compensation Manually 81 Table of Contents 3 4 Chapter 5 Analysis Adding a Plot cironi 83 Editi PIOUS iernii aa 84 Changin FIO Ty penere E A S 85 Dophecatn Sa Plo besonnen T 85 Removime a Plot erani ote ee ee eee 85 Chanoine AXIS Scale Sarrance E REN 85 Changing
234. nears the top of the filter Make sure that the air release port is tightened and that there is no leakage of fluid If the air release port is overtightened the filter may become damaged Do not apply excess force when tightening 7 Tap the side of the sheath filter with your hand 2 or 3 times This dislodges any trapped air in the lines which then collects in the sheath filter A a cS 2 or 3 times Releasing Air in the Sheath Filter and Sorting Chip soueuslUle g Ja deyuy I 181 soueuajUley g Jajdeyy 182 Use the following procedure if there are air bubbles in the sheath filter Open the air release port to expel the trapped air 2 Check that the air has been released and then close the air release port 3 Tap the filter again and check that there are no air bubbles being formed Repeat steps G to until all air has been released 8 Place the sheath filter in the retaining clamp then close the fluidics maintenance door 9 Click Ready in the Pressure Options tab of the Advanced Settings dialog The sheath fluid flow restarts Check that there are no air bubbles in the sheath filter before running samples for measurement It is recommended that you use this procedure if there are any air bubbles in the sheath filter Releasing Air in the DI Water Filter Releasing Air in the DI Water Filter During operation air may become trapped in the DI water filter interru
235. ng an Experiment When data acquisition for an experiment is finished the data for the experiment can be exported to an external file 1 Click Database on the File tab of the ribbon then click Export The Export Experiment Data dialog appears Experiment List Name FAEERE 2 Select the owner of the data in User and select to export experiments or templates using the Type radio buttons 3 Select an experiment you want to export in Experiment List and click gt gt to move it to Selected Experiment List Clicking All gt gt moves all the experiments to Selected Experiment List e To remove the exported experiments from the Experiment Explorer after the export is finished place a check mark in Delete selected experiments after export completed e To open the export destination folder after the export is finished place a check mark in Open exported folder after export completed A Click Browse to specify the destination folder in which the experiment data is to be exported Exporting Importing an Experiment 139 sluewuedxy Buunbyuoy p Jaideyo HIM s yu wn dx Buunbyuog p Je deyD 76 5 Click Export The export status is displayed in the progress bar A confirmation dialog appears when exporting 1s completed 6 Click OK Click Close to close the dialog Importing an Experiment You can import previously exported experiment data into the da
236. ng function If the volume of fluid is insufficient the cleaning fluids may be ejected from the sorting chip in a spray contaminating the optical lenses and other components General Cleaning The sorting area and sample loader should be cleaned regularly to prevent the precipitation of salts from the sheath fluid and to prevent the spread of biological matter Cleaning the instrument regularly also prevents trouble in the cultivation process after sorting Clean the droplet camera window laser windows collection stage collection tube holders and sample loader using water and ethanol e Be aware of the door edges when doors are open e Always wear gloves and other protective clothing mask and goggles 1 Tum on the power supply using the POWER STANDBY button on the front panel 2 Press the OPEN CLOSE sample loader door button on the front panel to access the sample loader 3 Press the PUSH OPEN sorting area door button on the front panel to access the sorting area 4 Moisten a clean soft lint free laboratory use cloth with clean water and wipe with the cloth Droplet camera window and laser windows Clean the droplet camera window to prevent reflections entering the camera and the laser windows to ensure the detection of the stream position If the windows become dirty an error may occur during automatic calibration Tip The number of laser windows varies depending on the model General Cleaning soueuslUl
237. ng stops automatically after satisfying an internal record stop condition when sufficient data has been recorded Tip You can stop data acquisition and recording manually using Stop if a sufficient number of events have been recorded and the plot view has stabilized 11 Click Next Compensation Wizard lt lt Acquisition has completed Verify the position of the v 1 Gain Settings catter gate and en e fluoresc mpass the negative events Select Next button to continue Negative control Data acquisition and recording for the negative control tube is completed You are then prompted to load and record the positive control tubes 12 Load the next control tube displayed in the wizard then click Start on the data acquisition control pane Status Ready Elapsed Time 00 00 00 Total Event 0 Event Rate g eps po E Start The positive controls must be loaded in the sequence displayed in the Experiment Explorer as prompted by the Compensation Wizard 13 Check that data acquisition has started then click Record in the data acquisition control pane Record Restart Pause Stop Recording stops automatically after satisfying an internal record stop condition when sufficient data has been recorded The positive population 1s displayed on the histogram plot Tip You can stop data acquisition and recording manually using Stop if a sufficient number of events have been recorded and
238. ngth for each model see Fluorochrome Detection Matrix page 204 e The 488 nm excitation laser is used to measure forward scatter and back scatter light e It is recommended that lasers not required for an experiment be deselected e The 561 nm excitation laser takes approximately ten minutes to reach full working temperature The Filter Setting screen appears Initial Instrument Setup Insert all the optical filters in the slots specified in Fluorochrome Detection Matrix page 204 and close the flip up door then click Next Jnitial Instrument Setup Vv 1 Chip Detection Please check filter setting first then close the upper door vV 2 Chip Information vV 3 Chip Loading Vv 4 Laser Setting 5 Filter Setting 6 Fluidics Check The image displayed shows the optical filter pattern to be installed The emission spectra of the fluorochrome labels you wish to detect determine the optical filters needed for an experiment For a list of fluorochromes versus optical filters and the corresponding optical filter channels for each model see Fluorochrome Detection Matrix page 204 Make sure that the correct filter is inserted in each slot for the recommended filter pattern Correct data cannot be obtained if a filter is inserted in the wrong slot Exercise extreme care when handling the optical filters The surfaces of each optical filter must be free of dust smudges fingerprints a
239. not guaranteed if an external device is connected CAUTION For safety do not connect the connector to any peripheral device wiring that might apply a high voltage to this port Name and Function of Parts Follow the instructions for this port Heat exhaust vent Discharges heat generated from inside the unit AMS evacuator adapter connection point Use a specialized adapter to connect a S A F E System evacuator from Edge Systems LLC For details see Connecting an AMS Evacuator SHSOOZ and SHS00ZP page 213 Maintain at least 10 cm 4 in space behind the main unit to allow for the dissipation of heat and to provide access to the power connector main power switch electrical connectors fluidics cart connectors and compressed air supply connectors 1 Compressed air supply fluidics cart connector block SHEATH Cart connection cable connector CART Connects to the cart connection cable connector on the fluidics cart It is used to connect the sensors for the three fluid tanks in the fluidics cart Use only the supplied cable to connect the main unit to the fluidics cart Using third party cables may cause incorrect operation or device failure Fluidics cart Waste tank Ethanol tank Sheath tank i Adjustable feet Tray Waste tank Collects the waste fluid comprising the sample fluid and sheath fluid pumped from various points within the main unit It also c
240. nt Explorer Show Results Displays the Tube Results dialog page 115 for displaying the recording results and sorting results for the tube selected in the Experiment Explorer If 8 Well Strips Slides 96 Well Plate or 384 Well Plate sorting was performed the data source results that were recorded last are also displayed If you click this button while a data source is selected the Data Source Results dialog page 117 appears allowing you to view the results of each data source Export group Contains a tool button for exporting recorded data for a sample tube FCS Export to FCS file r Export to FCS Files Displays the FCS File Export dialog page 117 for exporting the data recorded for the tubes in the experiment selected in the Experiment Explorer in FCS 3 0 or FCS 3 1 format Tip The importing of FCS files is not supported in SH800 Software Save as Template dialog The Save as Template dialog is displayed by selecting an experiment sample group or sample tube in the Experiment Explorer and clicking Save as Template on the Experiment tab of the ribbon or by right clicking and selecting Save as Template from the context menu The Save as Template dialog is used to save the selected experiment sample group or sample tube as a template Save as Template Template Section My Templates Y Template Name For details see Saving an Experiment as a Template page 78
241. ntly wipe the surface using lens paper Use a fresh surface of the cloth for each wipe Maintain a continuous wiping motion at a constant speed until the cloth passes the edge of the optical filter 4 After cleaning insert the optical filter into the slot and push the lip of the optical filter until it clicks into place e Avoid touching or wiping coated or metal mirror surfaces at all times except when cleaning e Avoid handling exposed coatings with bare fingers e Always wear gloves and also wear a face mask goggles and other protective clothing as required to prevent oils from your hands contaminating the optical glass and to prevent solvents and chemicals coming into contact with your skin or eyes Disconnecting and Reconnecting the Fluidics Cart After installation it may be necessary to disconnect and then reconnect the fluidics cart if you wish to move the fluidics cart and need to re route the path of the fluidics lines sheath air line and cart connection cable between the fluidics cart and the main unit If you wish to move the SH800 main unit contact your Sony distributor Do not attempt to relocate it yourself The main unit contains high precision optical and laser instruments that require alignment after installation that only your Sony distributor can provide 1 With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel of the main unit 2 Disconnect t
242. o Settings Only Hardware Cleaning SAL TEELORNTI meig The Shutdown Wizard appears A Click Start Shutdown Wizard To keep cytometer condition please do belach cleaning and DI rinse before shutting down Click Start button to initiate shutdown procedure If you need to shutdown imediately please ckick Shutdown button Tip If you click Shutdown the SH800 shuts down without doing any cleaning 5 Select the sodium hypochlorite cleaning sample tube size and click Start Shutdown Wizard 1 Bleach cleaning Please select cleaning mode and load the selected size of centrifuge tube Then click Start button to initiate bleach cleaning procidure If you don t need click Abort button Carefull cleaning 30ml centrifuge tube with 25ml of sodium hypochiorite solution Bleach cleaning starts When bleach cleaning finishes flush the system using DI water 6 Select the DI water cleaning sample tube size and click Start To shutdown the instrument with the sample probe filled with DI water place a check mark in Keep DI water inside sample probe Shutdown Wizard 1 Bleach cleaning Please select rinse mode and load the selected size of centrifuge tube Then click Start button to initiate DI rinse procidure 2 DI water rinse If you don t need click Abort button O Normal cleaning Normal cleaning 15ml centrifuge tube with 12ml of sodium 5 Carefull cleaning
243. of the sample tube required item Date Displays the date the sample tube was created This parameter cannot be modified Sample ID1 to Sample ID4 ID labels for identifying the sample tube OK Applies the settings and closes the dialog Cancel Cancels settings and closes the dialog Apply Applies the settings without closing the dialog Worksheet Settings dialog The Worksheet Settings dialog is displayed by double clicking Worksheet Settings for a sample tube in the Experiment Explorer The Worksheet Settings dialog is used to save the current worksheet settings number of plots type of plots statistics settings etc as the favorite settings Saving current worksheet settings as the favorite settings allows you to restore the settings at a later time or to apply the settings to a different sample tube Tip Data that is acquired and recorded for a tube is not included in the worksheet settings Tube 1 Worksheet Settings Current Worksheet Settings Modified 2 23 2014 4 28 27 PM Save as Favorite Restore Favorite Favorite Worksheet Settings Modified 2 23 2014 4 58 53 PM Preview Current Worksheet Settings Modified Displays the date and time that the current worksheet settings were modified Restore Favorite Restores the saved favorite worksheet settings as the current worksheet settings Save as Favorite Saves the current worksheet settings as the favorite defau
244. ollects ethanol and DI water during cleaning A sensor detects the waste fluid level for display on the LCD monitor and host computer An Compressed air supply input connector AIR IN Connects to the laboratory compressed air supply or a standalone air compressor Fluidics cart air line connector AIR OUT Connects to the sheath air line connector on the fluidics cart clear tubing The air line supplies regulated compressed air for the sheath tank Fluidics cart sheath line blue connector Connects to the SHEATH connector on the fluidics cart using the supplied sheath line blue tubing Fluidics cart ethanol line green connector Connects to the ETHANOL connector on the fluidics cart using the supplied ethanol line green tubing Fluidics cart waste fluid line red connector Connects to the WASTE connector on the fluidics cart using the supplied waste line red tubing The standard fluidics lines sheath air line and connection cable are about 2 m 6 ft in length It is recommended that the SH800 be placed on a table of height approximately 700 mm 28 in or greater and that the fluidics cart be placed on the floor Cart connection cable connector MAIN UNIT a 1 Air line fluidics line connector block alarm is generated and SH800 operation automatically stops when the waste fluid rises above a set level The tank has a capacity of 10 liters 2 6 gallons US Name and Function of Parts
245. omize the structure of an experiment created using templates or recent experiments by adding sample groups and sample tubes to the experiment and configuring the settings of each component You can also customize an experiment after it has been created For details see Editing an Experiment page 74 1 Click New on the File tab of the ribbon or click New in the Experiment group on the Experiment tab of the ribbon The Create Experiment window appears 2 Selecta template or a recent experiment Clicking a template or an experiment on the left displays the structure of the selected experiment on the right side of the window 70 Creating a Customized Experiment 3 Modify the structure of the experiment as desired Blank Template HS h iment 12 28 20 3 8365 01 PM ga Experiment Information E Sample Group 1 amp Sample Group Information Measurement Settings Compensation Settings 4 44 Compensation Panel Negative control Briliant Violet 421 FITC DE APC PerCP Cy5 5 PE Cy7 oe o m e OO LALLI Tube Information Worksheet Settings Stop Conditions amp Sort Mode For details about adding deleting sample groups and sample tubes see To add a sample group page 71 To add a sample tube page 72 and To delete a sample group or tube page 73 Tip Changes made to the settings on the right side of the window are immediately reflected in the
246. on the main unit It is used to connect the sensors for the three fluid tanks in the fluidics cart Use only the supplied cable to connect the main unit to the fluidics cart Using third party cables may cause incorrect operation or device failure The standard cable is about 2 m 6 ft in length 1 Air line fluidics line connector block AIRIN SHEATH ETHANOL WASTE rit Fluidics cart air line connector AIR IN Connects to the fluidics cart air line connector AIR OUT on the SH800 clear tubing The air line supplies regulated compressed air for the sheath tank The SH800 is designed to connect to 500 kPa to 650 kPa 73 psi to 94 psi compressed air supply for stable output Fluidics cart sheath line blue connector Connects to the SHEATH connector on the main unit using the supplied sheath line blue tubing Fluidics cart ethanol line green connector Connects to the ETHANOL connector on the main unit using the supplied ethanol line green tubing Fluidics cart waste fluid line red connector Connects to the WASTE connector on the main unit using the supplied waste line red tubing e The standard fluidics lines and cable are about 2 m 6 ft in length Use of lines longer than 2 m 6 ft may impair the pumping of fluids from the fluidics cart e Use only the supplied cable to connect the main unit to the fluidics cart Using third party cables may cause incorrect operation or device failure Sort
247. onent which can be damaged easily if handled carelessly Changing the PEEK Sample Line PEEK sample line compatible models 9 e The sample line can be discarded Discard the sample line in accordance with the laboratory rules and local ordinances and regulations Prepare a new PEEK sample line then click Next Insert the PEEK sample line into the probe adapter grasp the black fitting and gently screw it clockwise to secure it then click Next to be a Probe adapter w E Fully insert and tighten the threaded portion of the fitting If the fitting is not fully secured the correct sample pressure may not be applied Attach the new sample line to the inlet port on the front of the chip loader then click Next Screw part A until it clicks into place making sure it is securely attached When attaching the sample line always hold part A of the connector not the T shaped portion Holding the connector by the T shaped portion may cause tightening that exceeds the appropriate tightening torque and adversely affect instrument performance e When attaching the sample line connector always screw in the connector until you hear a clicking sound e Check the sample line connector regularly for any signs of looseness 1 0 Check that the sample line connector is attached correctly then click Next When installed correctly the stepped portion of the sample line connector fits under the cover
248. or any claims from a third party arising out of use of the unit or supplied software For complete terms and conditions of the warranty for the unit refer to the warranty card included in the package The software supplied with the unit cannot be used with any other units Note that the specifications of the unit and supplied software are subject to change for improvement without prior notice Table of Contents 8 Software Revision History Functions Added in Version 1 7 The following functions have been added in SH800 Software version 1 7 Support for PEEK sample line Support added for PEEK sample line that can be detached easily and safely For details see Sample Line Support page 10 Support for 384 well plates option Sample fluid can now be sorted into 384 well plates using this option For details see Preparing Collection Tubes page 33 and Sorting page 53 Added sample line exchange to maintenance mode The sample line can be replaced using a wizard when the unit 1s in maintenance mode For details see Running Maintenance Mode page 158 Support for sample line cleaning at startup Sample line cleaning can be performed during initial setup when starting SH800 Software For details see Initial Instrument Setup page 43 Improved functionality The following functions have also been added or expanded e Display of remaining sorting time page 136
249. ormed or the The droplet camera may be defective Contact your Sony distributor for service droplet camera image is not displayed Data is not acquired in a given The optical filters may not be inserted in the correct filter pattern or may require fluorescence detection channel or cleaning Check that the filters are clean and inserted in the correct slots condition less than favorable Gain settings appear to have deviated The optical filters may have been subjected to static electricity discharge Pause during measurement or stop data acquisition and then resume data acquisition to restore normal gain settings No output from the lasers laser If the ambient operating temperature exceeds the maximum operating temperature error temperature of 35 C 95 F the lasers shutdown automatically and an error message is displayed To clear the error shut down the main unit and wait for the ambient temperature to cool sufficiently before restarting the main unit If the error persists even after allowing the ambient temperature to cool below 35 C 95 F contact your Sony distributor for service If the ambient temperature drops below the minimum operating temperature of 10 C 50 F the lasers shut down automatically and an error message is displayed To clear the error shut down the main unit and wait for the ambient temperature to rise above 10 C 50 F before restarting the main unit If the error persists even after the tem
250. ount privileges e The screenshots displayed in this document may vary from the actual software 25 Name and Function of Parts roneo va WN MAIMOAQ Je deyD 26 Operating window Worksheet Tool Tube 2 index 96 Well Data Source 1 ie le A a Auto Adjust Auto Ad st Zi fat All Events Search Results From 2014 01 06 public 0 useri 6 E Experiment 2014 01 21 15 28 32 xperiment 2014 01 21 15 00 11 xperiment 2014 01 16 18 19 18 xperiment 2014 01 16 16 15 11 xperiment 2014 01 16 14 57 40 eak Index Test amp Experiment Information 4 ia Sample Group 1 Tube 1 2 4 Tube 2 1 Tube information gt Worksheet Settings Stop amp Sort Settings zi ndex 96 Welll Data Sou Tube 3 3 4 D Sample Group 2 10 10 10 FITC A Compensated Sorting Method 2 Way Tubes O A Ww se Polygon Quadrant 2 g to 10 FITC A Compensated 10 104 1 PE Cy A Compensated For details about the items displayed see Chapter 7 Window Description page 104 Ribbon Displays the tool buttons that can be used in the displayed window The content of the ribbon varies depending on the selected tab Worksheet Work area for displaying and editing the results from data acquisition and analysis Ribbon show hide control minimize restore Clicking 3 alternately shows the ribbon or hides the ribbon Logout Logs out the currently logged in u
251. p Sobeod gt Sodeod DOODO x er ft however there is a trade off to be made between yield recovery and purity Sorting is not an exact science There are several factors that can affect the outcome The position of a cell within a droplet can only be determined within a certain degree of accuracy fluctuations can occur in the fluid flow due to the passage of large cells or variations in the sheath fluid pressure and the droplet charging pulse timing may be slightly out of phase with droplet formation If a targeted event appears close to the top or bottom edge of a droplet the probability of correct recovery is reduced The targeted event may actually be output in an adjacent droplet One option is to sort successive droplets in order to catch the event to increase yield and recovery If a targeted event appears close to the edge of a droplet and an unwanted event occurs in the adjacent droplet or vice versa the decision whether to sort the droplets or not depends on the goal isolating a pure population or recovering as many targeted cells as possible Sorting the targeted event will increase the yield but with a subsequent reduction in purity Conversely rejecting the targeted event will maintain high purity but with reduced yield Likewise a similar decision occurs if a targeted event and an unwanted event appear in the same droplet e If two different targeted events for collection in separate collection tubes a
252. p Condition None Y 2 Place the collection tubes into the corresponding holder For details see Preparing Collection Tubes P i page 33 F Sorting 53 uoneiedo oiseg g13 deyo Kii uoneledg siseg Jaldeuy 54 SH800ZP 6 Set each parameter For details about each item see Sorting Control Pane page 136 w Sort Control A i gt Sort Start Unload Collection R Sorting Method 2 Way Tubes ToSort A v A v Mode Normal v Regular Cell Stop Value 0 0 Mode Selects the sorting mode To Sort Selects the gates for the target events to sort into the left and right collection tubes Stop Value Specifies the event count conditions to stop recording automatically for each of the left and right collection tubes You can select a value or enter a value from the keyboard Entering a value of 0 disables the stop condition T Click Start or Resume in the data acquisition 4 Click Load Collection in the Sort Control pane control pane to begin data acquisition status Ready Elapsed Time 00 00 00 Total Event 0 y Sort Control Event Rate 0 eps Sorting Method 2 Way Tubes 7 m m Made Normal x Regular Cell 7 The collection tube device is loaded 8 Click Sort Start in the Sort Control pane to begin Note sorting Do not open the sorting area door during sorting Y Sort Control Select 2
253. p the instrument page 43 Configure the required settings for data acquisition Run automatic calibration page 46 Run automatic calibration to align the sorting chip and calibrate droplet formation using automatic setup beads 4 4 Step 5 Create an experiment page 47 Step 6 Acquire data page 49 Analyze data page 51 Sorting page 53 Clean fluidics system page 143 Clean the sample probe sample line and sheath line Shut down page 65 Shut down the main unit and clean the sample loader and sorting area System Startup Turn on the power supply and start the SH800 using the following procedure Do not open the flip up door at any time before turning on the compressed air supply Doing so may damage the unit 1 2 Check that the compressed air supply is connected and is supplying the rated pressure Check the level in the tanks on the fluidics cart and refill or empty the tanks as required Also check that the fluidics lines sheath air line and cart connection cable are connected correctly Turn on the MAIN POWER switch on the rear panel of the main unit During normal operation the MAIN POWER switch can be left turned on all the time Use the POWER STANDBY button to turn power on off on a daily basis Turn on the power using the POWER STANDBY button on the front panel of the main unit The system goes through an initialization process during which self dia
254. packaging for the loaded sorting chip should be stored in a safe location e Replace the sorting chip if it is discolored dirty or stained from previous use e Do not drop the sorting chip on a hard surface or bend the chip intentionally If there is a problem with the sorting chip contact your Sony distributor Initial Instrument Setup 43 uoneiedo oiseg g13 deyo Kii uoleisdo olseg Jsa deuyD O e Always wear gloves and other protective clothing mask and goggles when changing the sorting chip If the sorting chip will not load correctly run the instrument in maintenance mode as described in Running Maintenance Mode page 158 to clean the instrument If the problem persists contact your Sony distributor for service Replace the sorting if it is used for extended periods or after the usage period expires and when it becomes clogged discolored or stained Click Next The sorting chip is loaded and the Laser Setting screen appears Select the excitation lasers to be used for the experiment then click Next Initial Instrument Setup Vv 1 Chip Detection Please select laser s that you will use for the experiment vV 2 Chip Information vV 3 Chip Loading 4 Laser Setting 405nm 488nm TESH The excitation spectra of the fluorochrome labels you wish to detect largely determine the lasers needed for an experiment For a list of fluorochromes versus excitation laser wavele
255. perature has risen above 10 C 50 F contact your Sony distributor for service Expected data is not acquired using Check the beads lot and dilution concentration reference beads Waste fluid is not being collected inthe Waste fluid may be leaking in the fluidics cart due to an unsecured waste line waste tank connector on the waste tank Make sure the waste line connector is securely connected on the waste tank Replace the waste tank air filter if more than one month has elapsed since the air filter was last replaced If the problem persists contact your Sony distributor for service Communication error between the Shutdown and restart both the SH800 main unit and the host computer SH800 and the host computer POWER STANDBY button indicator is If the doors can be opened and closed remove the sample tube and collection flashing tubes before performing the following procedure 1 Press and hold the POWER STANDBY button until the indicator stops flashing 2 Press the POWER STANDBY button again to start the main unit If the POWER STANDBY button indicator does not flash continue normal operation 3 If the POWER STANDBY button indicator starts flashing again press and hold the POWER STANDBY button until the indicator stops flashing then turn off the main power switch on the rear panel 4 Contact your Sony distributor for service Troubleshooting Possible Cause Recommended Solution An error occurs in automatic c
256. ple Turns the sample loader light on off Cleaning group Contains tool buttons for cleaning the fluidics system i i O T s HCI Di CN Probe Bleach Dy Shutdown Wash Cleaning Rinse Rinse ean Probe Wash Washes the sorting chip sample line and the inner and outer surfaces of the sample probe using sheath fluid Bleach Cleaning Displays the Bleach Cleaning dialog page 120 for cleaning the sorting chip sample line and sample probe with a Sodium Hypochlorite solution For details about cleaning using Sodium Hypochlorite solution see Cleaning the Sample Fluidics System using Bleach page 152 DI Rinse Displays the DI Rinse dialog page 120 for flushing the sorting chip sample line and sample probe with DI water Shutdown Rinse Displays the Shutdown DI Rinse dialog page 120 for flushing the sorting chip sample line and sample probe with DI water before shutting down the instrument After cleaning the fluidics lines are filled with DI water to prevent the formation of salt deposits Running data acquisition in this state may damage cells De bubble group Contains tool buttons for removing air bubbles from the sorting chip and sheath filter F i 3 Chip Sheath Fitter le bubbie Chip Removes air bubbles from the sorting chip Main Window Sheath Filter Displays the Sheath Filter De bubble dialog page 120 for removing air bubbles from the she
257. plicates the selected plot on the worksheet Main Window uonduos q mopuiM Z JeldeyyD I 125 uonduoseg MOpUIN Z Je deuyD 126 Display Events group Contains tool buttons for setting the number of events displayed on plots ES Acquisition 5 000 H Analysis 100000 oe r m Hens siere 5p a g Acquisition Sets the number of events to display on plot during data acquisition Analysis Sets the number of events to display on plot when analyzing data Gate group Contains tool buttons for editing gates GR Remove Edit Gate Gate For details see Editing Gates page 91 Remove Gate Deletes the selected gate Deleting a gate will also delete all gates and plots that are derived from the selected gate Edit Gate Displays the Gate Editor dialog page 127 for editing gates Statistics group Contains tool buttons for working with statistics y ms HHA J Show Edit Export Statistics Tabie Statistics to CSV File Statistics For details see Displaying Statistics page 92 Show Table Shows hides the Gates and Statistics table Edit Statistics Displays the Statistics Editor dialog for configuring the attributes displayed in the Gates and Statistics table For details about the Statistics Editor dialog see Displaying Statistics page 92 Export Statistics to CSV File Displays the Exporting statistics table
258. ppear in the same droplet or close to one another in adjacent droplets the sorting decision must determine which of the two targeted events is sorted correctly or whether to abort sorting of the two events e If two or more cells including target and or unwanted cells appear at the optical detection point in very close proximity a single event will be triggered if the detected signal does not fall below the threshold level between cells In this case it is impossible to distinguish the event as separate cells in a single pass sort If the event contains targeted and unwanted cells it will exhibit some of the characteristics of both types of cell If the sort criteria only define a limited subset of targeted cell characteristics the event may satisfy that criteria and be sorted accordingly with a corresponding reduction in purity due to the presence of the unwanted event Sorting using multiple gates to isolate target cells for multiple characteristics can help isolate targeted events In addition hardware related factors can affect sorting If two or more cells including target and or unwanted cells appear at the optical detection point in reasonably close proximity separate events will be triggered if the detected signal falls below the threshold level between cells However if the acquisition electronics are not yet ready to receive the second event the physical presence of the second event is detected but the event is not analyz
259. pting the smooth flow of DI water To release air trapped in the DI water filter using the de bubble function 1 Click Settings on the Cytometer tab of the ribbon The Cytometer Settings dialog appears 2 Click DI Water de bubble on the Maintenance tab The DI Water de bubble dialog appears 3 Click Start 4 Open the fluidics maintenance door remove the DI water filter from the retaining clamp without disconnecting the connectors 5 Slowly open the air release port on the top of the DI water filter e It is recommended that the drip tray be pulled out partway before proceeding Slowly open the air release port while holding a soft clean cloth ready to wipe away any DI water overflow e Keep the DI water filter clear of all other objects when the air release port is open The trapped air escapes and the fluid level in the DI water filter begins to rise Close the air release port when the fluid level nears the top of the filter Make sure that the air release port is tightened and that there is no leakage of fluid If the air release port is overtightened the filter may become damaged Do not apply excess force when tightening Click Stop Place the DI water filter in the retaining clamp and close the fluidics maintenance door When finished click Close Autoclaving the Sheath Filter and DI Water Filter You can autoclave the sheath filter and DI water filter if using autoclavable
260. r operator PROJ Name of the experiment project SMNO Specimen e g tube label SRC Source of the specimen patient name cell type CELLS Description of objects measured SCOM Saving an Experiment as a Template Saving an Experiment as a Template You can save an experiment sample group or sample tube as a shared template Public Templates or a private template My Templates 1 Select the target experiment in the Experiment Explorer on the Experiments tab on the left of the main screen Click Save as Template in the Experiment group on the Experiment tab of the ribbon j si l a Experiment Cytometer Compensation g gt Ea x fis Next New Up O New Tube The Save as Template dialog appears Select the template type enter a name and then click OK Save as Template Template Section My Templates v Template Name The experiment is saved as a template Configuring Detector Settings The following instrument settings must be optimized in an experiment to acquire accurate data e Trigger threshold channel e Trigger threshold value e Sensor gain of the forward scatter back scatter and fluorescence detectors The instrument settings are saved with the sample group in an experiment Duplicating an experiment or creating a new experiment using templates will load the saved instrument settings into the new experiment Running an experiment using the same ins
261. r supply to the main unit and start SH800 Software 1 1 Click Probe Wash on the Cytometer tab of the ribbon to wash the sample probe Repeat ten times to fill the DI water line with ethanol 12 Let stand for 20 minutes then remove the cap of the DI water tank and dispose of the ethanol Dispose of ethanol in accordance with laboratory rules and with local and federal ordinances and regulations 1 3 Fill the DI water tank with DI water then shake the tank with your finger over the air filter connection port to rinse the tank 14 In SH800 Software click Probe Wash on the Cytometer tab of the ribbon to wash the sample probe Repeat ten times to remove all traces of ethanol from the DI water line 15 Remove the cap from the DI water tank and dispose of the DI water Dispose of DI water in accordance with laboratory rules and with local and federal ordinances and regulations 1 6 Connect the quick release connector to the air filter Cleaning the DI Water Tank and DI Water Line 17 Refill the tank with fresh DI water up to the opening of the tank and reattach the cap 1 8 Re install the DI water tank Install in the reverse order of removal 1 9 Disconnect the DI water filter bypass line green and reattach the DI water filter 20 Check the drip tray for any fluid spills or leakages and clean as necessary 21 Close the door and turn on the power supply 22 In SH800 Software click Probe Wash
262. rces are scarce because of multiple Close any unnecessary running applications and processes applications processes running in the background This may impact the performance of the SH800 Software Please close unnecessary applications processes Invalid biexponential parameters were selected Specify valid biexponential parameters Cannot assign this tube because available free size of HDD Cannot assign due to insufficient remaining space on HDD is not enough Please exceed available free size space of HDD the hard Delete unnecessary files to increase the available free capacity drive Detected UNKNOWN state Please check the cytometer Check the cytometer for any abnormalities SH800 Unable to maintain stable droplet breakoff The measurement Data acquisition stops Check the instrument settings as will be stopped and Control Breakoff will be disabled required This chip has been used over 9 hours with the 405 nm laser The sorting chip is approaching the end of its service life the limit with a 405 nm laser is 12 hours Please exchange Replace the sorting chip the chip before the 12 hour limit Cannot receive droplet camera image continuously If you The droplet camera image cannot be acquired If a problem have some trouble please stop acquisition occurs stop data acquisition Cannot receive side stream camera image continuously If The side stream monitor image cannot be acquired If a you have some troub
263. rding to the schedule below Preparations for the Day Before Measurement Maintenance item Refilling the e Itis Refilling the sheath tank recommended Sheath Tank to fill the tank page 146 the day before operation to allow air bubbles to escape Maintenance for Each Startup Operation Maintenance item Disposing of waste fluid Replacing the waste tank Check that the tank is not full when starting up and shutting Emptying Changing the Waste Tank page 148 down Replace when nearly full or when prompted in SH800 software Checking that When starting there is sufficient up volume in the sheath tank Refilling the sheath tank Refilling the Sheath Tank page 146 When prompted in SH800 Software Refilling the DI water tank up When prompted in SH800 Software As required When starting Refilling the DI Water Tank page 149 Maintenance for Each Shutdown Operation Sample fluidics system cleaning Maintenance item Sample fluidics e When shutting Cleaning the down at day s Sample Fluidics end System using e As required Bleach page 152 system cleaning Maintenance Schedule soueUuslUle y g Jaideyy I 143 Tip Periodic Maintenance Use the same procedure above to clean the sample line Maintenance item and sample probe even on models that do not support the Changing the Every 3 months Changing the
264. re detected during sorting in units of events per second eps Sort Efficiency Displays the number of sorts attempted as a percentage of targeted cells in the sample Abort Count Displays the number of events that do not match the sort criteria and which are aborted during sorting Abort Rate Displays the rate at which events are aborted during sorting in units of events per second eps Droplet Viewer window The Droplet Viewer window is displayed by double clicking the image in the Droplet pane at the bottom of the main window The Droplet Viewer window is used to monitor droplet formation and as a guide when adjusting the breakoff point Main Window Zoom factor Selects the zoom factor lt 1 00 0 75 or x0 50 Status icon B Indicates the stability of droplet formation Indication Status Flashing slowly Waiting for stream to stabilize Flashing rapidly Adjustment in progress ty Indicates that the droplet control breakoff function is active No icon Indicates unstable or absence of droplet formation Droplet monitor Displays the droplet stream Control Breakoff The control breakoff setting uses a feedback system to control the breakoff position automatically When enabled the amplitude and frequency settings are adjusted automatically to maintain a stable breakoff point position with coherent side streams When adjusting the breakoff point first set the amplit
265. reate Parent Plot Adds a parent plot to the worksheet Copy Selected Gate Copies the selected gate Context Menu uonduos q MOpUIAA Z JeldeuD I 141 uogdoseq mopuiM Z Je deyD 142 Cut Selected Gate Cuts the selected gate Paste Selected Gate Pastes a previously copied or cut gate Visible Gate Shows hides the selected gate Remove Selected Gate Deletes the selected gate Show Table Shows hides the Gates and Statistics table Save as CSV File Displays the Exporting statistics table dialog page 128 for exporting the data in the Gates and Statistics table as a CSV format file Open Gate Editor Displays the Gate Editor dialog page 127 to edit gates Open Statistics Editor Displays the Statistics Editor dialog page 92 to configure the statistics displayed in the Gates and Statistics table Refresh List Updates the content displayed in the Gates and Statistics table Properties Displays the Property Window dialog page 130 Context Menu Maintenance Chapter This chapter describes the routine maintenance procedures required to maintain accurate performance and longevity of the SH800 Fluids may contain biological chemical or other agents Always wear gloves and other protective clothing mask and goggles as required when performing maintenance Maintenance Schedule The maintenance procedures outlined in this manual should be performed acco
266. red Every 3 to 12 months As required Every 3 to 6 months Every 12 months Every 12 months Every 12 months Every 12 months Every 12 months Every 12 months PEEK Sample Line PEEK sample line compatible models page 169 Changing the Sample Line and Probe Non PEEK sample line compatible models page 172 Changing the Sheath Filter page 176 Changing the DI Water Filter page 177 Changing the Sample Loader O Ring page 160 Changing the Sample Line and Probe Non PEEK sample line compatible models page 172 Changing the Probe Adapter PEEK sample line compatible models page 175 Changing the Sintered Sheath Line Filter page 179 Changing Tank Air Filters page 165 Cleaning the Waste Catcher page 157 Miscellaneous e As required Cleaning the when dirty Deflection Plates page 155 Cleaning the Waste Catcher page 157 Running Waste Line A Maintenance item Cleaning the deflection plates Cleaning the waste catcher As required when dirty After a week or more of non use Maintenance e As required page 159 As required Changing the Sample Loader O Ring page 160 Running Waste Line A maintenance Cleaning the sample loader O ring Cleaning the sheath tank and sheath line As required Cleaning the Sheath Tank
267. reen appears before exiting the Sample Line Exchange wizard Reinsert the unloaded sorting chip then click Next When the sample line has been changed the chip exchange screen appears Changing the Sample Line and Probe Non PEEK sample line compatible models Changing the Probe Adapter PEEK sample line compatible models The probe adapter that accepts the probe part of the PEEK sample line must be replaced regularly The probe adapter can be removed disinfected using autoclaving and then reinserted back into the unit Sample fluid may contain biological chemical or other agents Always wear biology laboratory gloves and other protective clothing mask and goggles when changing the probe adapter Change the probe adapter using the following procedure after removing the PEEK sample line In particular when disinfecting the probe adapter for reuse always follow the procedure correctly to change the adapter Remove the PEEK sample line For details about removal see steps 1 to 6in Changing the PEEK Sample Line PEEK sample line compatible models page 169 2 Remove the probe adapter Push and hold the sample probe release catch to the right then pull the probe straight up and out 3 Disinfect the probe adapter as required Autoclave the probe adapter under general autoclave conditions 121 C 250 F 2 atmospheres approx 20 minutes Make sure the probe adapter is autoclaved if reus
268. riment tab of the ribbon has the following buttons Target group Contains tool buttons for specifying the active sample tube used for data acquisition recording and sorting Assign Tube Assigns the tube selected in the Experiment Explorer as the active tube for data acquisition recording and cell sorting Next Tube Assigns the next tube If there are no further tubes it duplicates the current tube and assigns the new tube Main Window uonduos q mopuiM Z JeldeyD I 113 uondosaqg mopuiM Z Je deuyD 114 Experiment group Contains tool buttons for creating deleting and performing other actions on experiments z i rs 2 A el BA Hew New Displays the Create Experiment window page 111 for creating a new experiment Delete Deletes the experiment selected in the Experiment Explorer Duplicate Duplicates the experiment selected in the Experiment Explorer Save as Template Displays the Save as Template dialog page 115 for saving the experiment selected in the Experiment Explorer as a template used when creating a new experiment Send to Public Creates a duplicate of the selected experiment and places the duplicate under the Public node for sharing with other users Sample Group group Contains tool buttons for creating deleting and performing other actions on sample groups a n U nt Th aa 7 Ai 4 m 1 i Leite F a i ar J 4 Le we c mj
269. rix coefficients cannot be added or deleted Spillover Matrix Displays the fluorescence compensation spillover matrix Spillover matrix values display the fluorescence intensity due to each fluorochrome as a percentage of the total intensity in each channel Spillover matrix coefficients can be modified manually Changing compensation values directly is only recommended for experienced users Negative Value Displays the spillover matrix for the negative control tube for both area and height pulse parameters Save Saves the compensation settings as an XML file You can load saved compensation settings into another sample group or another experiment Load Loads previously saved compensation settings from an XML file Reset Resets all compensation settings Close Closes the dialog Calculate Compensation Settings dialog The Calculate Compensation Settings dialog is displayed by clicking Calculate Matrix on the Compensation tab of the ribbon The Calculate Compensation Settings dialog is used to calculate the coefficients of the fluorescence compensation spillover matrix based on the negative control tubes and the compensation panel Calculate Compensation Settings natrix will be calculated from compensation tubes data in compensation pane 7 Brilliant Violet 421 E B M Z vA o mc Hie Brilliant Violet421 10000 000 000 0 00 000 0 0 g PE is FC 0 00 100 00 0 00 000 000 0 00 amp arc His 5
270. rksheet The All Events plot is the top level in the hierarchy All plots for gates are derived from All Events If necessary adjust the axes scales using the tool buttons in the Axes group on the Plot Tools tab of the ribbon All Events 0 5 10 i5 20 25 EA 1 000 Adjust the gate size and position to surround the target population All Events x1 000 BSC A 0 5 10 15 20 25 ea x1 000 Double click the gate in the plot to add a child plot for that gate You can also add a child plot from the context menu by right clicking the gate in the Gates and Statistics table e You can add a new plot using the tool buttons in the Plot group page 125 on the Plot Tools tab or the Worksheet Tools tab of the ribbon e You can change the type of the plot using the tool buttons in the Plot Type group page 129 on the Plot Tools tab of the ribbon Click the axis labels and select the parameters to be displayed from the popup menu Adjust the axes scales to display the events on the plot as required A 1 000 BSC A 10 103 104 105 10 APC A Compensated Select a gate type in the Gate group of the Plot Tools tab and draw gates around both of the populations in the child plot The gates are automatically named in alphabetical order You can change the name and color of gates in the Property Window dialog by right clicking a gate and selecting Properties from the context menu
271. rns to its original position Deflection Control Contains controls for adjusting the side stream deflection angle loading unloading the collection stage and running a sorting test Stream Deflection Adjusts the angle of deflection of the two side streams Charge Peak Sets the peak value of the charging voltage waveform Collection Stage Loads unloads the collection stage Waste Catch Moves the sorting area waste catcher to the side Sort Test Runs a sorting test Sort Options tab The sort option settings are used to adjust the position of the sorting chip droplet camera and collection stage manually Sort Options Stream Options Pressure Options Chip Stage Main Window uondos q mopuiM Z JeldeyD I 123 uondosaqg mopuiM Z Je deuyD 124 Chip Stage Adjusts the positioning of the sorting chip within the chip loader Droplet Adjusts the vertical positioning of the droplet camera and the brightness of the droplet camera laser and backlight Collection Stage Adjusts the horizontal position of the collection stage Stream Options tab Adjusts side stream settings Sort Options Stream Options Pressure Options Side Stream Control Camera i Upper Position Camera Sets the viewing position upper or lower of the side stream monitor Side Stream LD Adjusts the horizontal position and power of the side stream monitor laser Deflection HV Adjus
272. rol Breakoff function Clicking this button displays a confirmation message Click Yes to save the current droplet image Control Breakoff The control breakoff setting uses a feedback system to control the breakoff position automatically When this function is enabled the drop drive is adjusted so as to maintain the optimized breakoff point and side streams obtained during auto calibration When auto calibration finishes the Control Breakoff function is automatically enabled The drop drive and drop clock cannot be adjusted manually while the Control Breakoff function is enabled When breakoff feedback control is enabled if the breakoff point moves and cannot be recovered within a predetermined time sorting stops and an error message is displayed on the screen Sample Empty Detection The empty detection function automatically stops data acquisition when the sample tube becomes empty The function detects disturbances in the droplet stream as the sample fluid in the sample tube is used up and then stops data acquisition e The sample empty detection function may stop data acquisition if cells of size 20 um or larger are in the sample If this occurs disable the sample empty function to continue data acquisition If a sample empty state is detected click Chip in the De bubble group on the Cytometer tab of the ribbon in SH800 Software and repeatedly de bubble the sorting chip until the breakoff point retu
273. rowth of bacteria With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel Refilling the DI Water Tank soueuslUle g Jaideyy I 149 soueuajUleW g sajdeyuy 150 2 Open the fluidics maintenance door then disconnect the line from the top of the DI water tank Press the metal release catch to withdraw the connector 3 Undo the metal restraining clamp and remove the DI water tank Refilling the DI Water Tank Always wear gloves and other protective clothing mask and goggles when handling fluidics system components Skin cells and hair are the most common cause of bacterial contaminations 4 5 Remove the cap and withdraw the tank probe Discard any remaining DI water Dispose of DI water in accordance with laboratory rules and with local and federal ordinances and regulations Refill the tank with fresh DI water up to the opening of the tank and reattach the cap Re install the DI water tank Install in the reverse order of removal Check the drip tray for any fluid spills or leakages and clean as necessary Q Close the door and turn on the power supply The DI water tank is fitted with an air filter that should be replaced every 12 months For details see Changing Tank Air Filters page 165 Refilling the Ethanol Tank The level of fluid in the ethanol tank can be monitored in the main window in SH800 Software and
274. ry and cell sorter operating principles of the SH800 Fluid Flow Sample injection chamber Waste tank fluidics cart Sample tube Sample fluid j y S A Pinch valve Deflection plates Side stream Appendix Sheath filter Sheath fluid I Charging electrode Main stream Waste catcher Side stream monitor point bon a Collection tubes Sheath tank fluidics cart Fluid Flow s jdi uld Dunesaodo y xipueddy 191 sojdiouudg Buryessdo y xipueddy 192 Sample Fluid Sample preparation is an important part of the testing procedure in flow cytometry Sample fluid must contain cells in a monodispersed suspension that has been filtered using an appropriate filter mesh to remove aggregates This is required to maintain a consistent sample concentration If the sample 1s not filtered the sample may clog or adhere to the surfaces of fluidics components and stable performance cannot be maintained For more information about sample preparation refer to reference sources Before running samples check that the sorting chip nozzle size 1s appropriate for the size of cells in the sample fluid As a generally accepted rule of thumb the nozzle size should be approximately five times min the diameter of the largest cell in the sample fluid to maintain stable sample fluid flow without clogging the chip The standard sorting chip has a 100 um nozzle which is sui
275. s Well Data Source 1 Plot Toots All Events A ty et Lh ite S o Swap Auto Adjust Auto Adjust Auto Adjust Z Zoom Default XY X Axis Y Axis Adjust Gate Tools Rectangle The mouse cursor changes to when the mouse is placed over the selected plot 2 Drag a selection rectangle over the portion of the plot to display then release the mouse PerCP Cy5 5 PerCP Cy5 5 All Events x1 000 The selected range is scaled to fill the plot area PerCP Cy5 5 PerCP Cy5 5 Tip Gates change shape on plots to track the scale type and zoom factor but may not be always reproduced accurately In particular calculation of polygon gates and ellipse gates are prone to error when a scale 1s changed from one type to another Editing Plots 87 se s10 sishjeuy g Je deyug Adding Gates You can isolate populations of events by drawing gates around the events on plots Gates of different types Rectangle Ellipse Polygon Quadrant Linear are available which can be drawn to match the distribution of events Adding a gate allows you to display a new plot representing the population of events that fall within the gate Gates are automatically added to the gate hierarchy in the Gates and Statistics table when they are added to plots You can also create Boolean gates which are defined by a logical combination of existing gates using Boolean logic Boolean gates are defined not by drawing ga
276. s 85 se so sishjeuy g Je deyug 86 For details about each item see Scale Type group page 129 You can select the following scale types Tip The axis scale type is applied only to the display of data on plots Internally all data is linear Linear Performs a linear transformation between the measurement values and the channel numbers bins used to measure data Linear scales are typically used to display forward scatter and back scatter parameters on plots 102 A a ee a 0 200 400 600 B00 CD56 Brilliant Violet 421 4 9 4 908 Log logarithmic Logarithmic log scales are useful for displaying cells that exhibit fluorescence with intensities across a wide dynamic range A logarithmic scale helps to make individual peaks easier to visualize However log scales cannot adequately represent data with a low mean and high variance 10 103 104 105 108 CD56 Brilliant Violet 421 A Biexponential Biexponential scales combine a linear scale at the lower end of the axis around zero and a logarithmic scale at the higher end of the axis with an algorithmic transition between the two scales A biexponential scale allows events close to or below zero to be plotted so that this population of events can be visualized making it easier to include or exclude populations when setting up gates 10 el 0 103 10 108 CD56 Brilliant Violet 421 A A common problem occurs when displaying fluorescence data on a lo
277. s 171 eoueuajUleyy g Jajdeyy 172 Changing the Sample Line and Probe Non PEEK sample line compatible models The sample line must be replaced regularly to prevent the buildup of salts restricting the sample fluid flow The sample line and probe can be removed independently or at the same time The sample probe can then be detached for autoclaving and then reattached to a replacement sample line for reinstallation The sample line should be checked regularly for any signs of damage or wear A damaged sample line may split and cause sample fluid leakage Always leave the sample line connected when the unit is not in use for protection against dust Use the following procedure to attach the sample line There is a risk of fluid leakage if the sample line is not attached correctly Sample fluid may contain biological chemical or other agents Always wear gloves and other protective clothing mask and goggles when changing the sample line or sample probe Sample line J i ma A mn L Sample probe 1 In SH800 Software click Sample line on the Cytometer tab of the ribbon 2 Click Start to begin changing the sample line and sample probe e Always follow the instructions displayed in the wizard when changing the sample line and sample probe e The sorting chip must be replaced or reinserted after changing the sample line and sample probe The sample line and sample probe can also be changed
278. s screw in the connector until you hear a clicking sound e Check the sample line connector regularly for any signs of looseness 10 Check that the sample line connector is attached correctly then click Next When installed correctly the stepped portion of the sample line connector fits under the cover View the connector from the side and check the stepped portion is not visible Check that stepped portion is not visible T T Correct Incorrect 11 Screw in the sample probe removing any twists in the sample line then click Next wba w _ vs ine Sample probe 12 Insert the sample line into the pinch valve then click Next Make sure the sample line is inserted all the way to the back of the pinch valve ee e If the sample line is not correctly inserted in the pinch valve sample fluid may leak from the probe Make sure the sample line connector 1s securely fastened and that the sample line is correctly inserted in the pinch valve If the sample line is not inserted correctly sheath fluid may flow in the reverse direction potentially causing serious damage to internal components and increasing the risk of biohazard contamination Be sure to insert the sample line into the pinch valve Failure to do so may result in problems caused by fluid leakage 13 Click OK to exit the Sample Line Exchange wizard When changing the sample line in maintenance mode the chip loading sc
279. s 176 Changing the DI Water Filter ccccssssessesseeeseeseesees 177 Changing the Sintered Sheath Line Filter 179 Table of Contents 5 Releasing Air in the Sheath Filter and Sorting Chip 181 Releasing Air in the DI Water Filter ccssscsssseeeeeees 182 Autoclaving the Sheath Filter and DI Water Filter 183 Ejecting the Sorting Chip Manually scsecessseseeees 185 Cleaning and Handling of Optical Filters 00 186 Disconnecting and Reconnecting the Fluidics Cart 187 Cleaning the Internal Sheath Line and DI Water Line using EMANO cece secsecsccedietacenseunict nieces wcadeeauenescaadiietvecateemecden 188 Appendix A Operating Principles PUGH FION jeeaeetceccegecataececati acteaenian ee atandi de cececasneus eecscseeeenceet 191 Sample FLUC sececons toss tinstiaxaieeetdaatactaceattietovstwnstiaxsietacnsntetoer aaeses 192 Bile ACTA FU o E IA PEIE EAN TEPE PITE OANT ETEA T 192 Hydrodynamic Focusing ccccccccssessssecceeeceeeeessseeeeeeeeeeeaaas 193 Droplet Sornos 194 OptiCal SYSTE sirsiran 196 Light and Signal Generation sssoosssooeesssssssssoerrsssssssseeerrsssss 196 Laser Interrogator sii ccveisecniccilicnszccnishardulocneeeeiasasivdhasceccsbseeadus 197 DE a EE AE E T EE S T EERTE tree OE 198 Fluorescence Compensation ccccccccccccessseeseeceeeeeeeeeaeeeeees 199 Appendix B Miscellaneous Sees LIN CUD acio
280. s of Life Technologies Corporation e eFluor is a registered trademark of eBioscience e Cy and CyDye are trademarks of GE Healthcare Ltd Brilliant Violet is a trademark of Sirigen Ltd Pacific Blue and Pacific Orange are trademarks of Life Technologies Corporation e S A F E System is a trademark of Edge Systems LLC The products or system names appearing in this document are trademarks or registered trademarks of their respective owners Further the or symbols are not used in the text http www sony net 2015 Sony Corporation
281. s the Export Experiment Data dialog page 109 for exporting experiment data You can export experiment data created on this unit for use by another SH800 Backup Displays the Backup Database dialog for backing up the experiment database This button is displayed for administrator accounts only Backing up the database allows the data to be restored at a later time if necessary For details see Backing Up Restoring the Database page 68 Restore Displays the Restore Database dialog for restoring a previously backed up experiment database This button is displayed for administrator accounts only For details see Backing Up Restoring the Database page 68 Delete All Data Displays the Delete All Data dialog page 109 for resetting the experiment database This button is displayed for administrator accounts only Resetting the database removes all data including user accounts experiments and analysis results Hard Disk Drive Capacity Displays the available free space on the hard disk A warning will appear when the free space on the hard disk falls below 50 GB The assigning of sample tubes is disabled when the free space falls below 20 GB Database Capacity Displays the available space on the database Up to 10 GB of data can be saved to the database in SH800 Software Various types of measurement conditions and configuration settings are saved in the database Measurement data is not
282. s them into the measurement settings for another sample group Show Settings Displays the Measurement Settings dialog page 73 for viewing measurement settings Context Menu uonduos q MOpUIAA Z JeldeuD I 139 uogdioseaq mopuiM Z Je deyD 140 Compensation settings The following menu commands are displayed when right clicking a Compensation Settings icon for a sample group in the Experiment Explorer Copy Paste Copies the compensation settings for the selected sample group and pastes them into the compensation settings for another sample group Show Settings Displays the Compensation Settings dialog page 124 for viewing compensation settings Sample tubes The following menu commands are displayed when right clicking a sample tube icon in the Experiment Explorer Assign Assigns the selected sample tube as the active tube for data acquisition recording and cell sorting Open Worksheet Opens the worksheet tab for the selected sample tube Apply Compensation Applies fluorescence compensation to the plots on the worksheet for the selected sample tube Copy Paste Copies the sample tube and pastes it into another sample tube Duplicate Duplicates the selected sample tube Save as Template Saves the selected sample tube as a private template Apply Template Displays the Apply Template dialog page 115 for selecting a template to apply to the selected sample tube
283. say tube sorting and 96 well plate sorting as examples Tip You can show hide the sorting control pane by clicking A in the top left corner of the pane In analyzer mode the sorting control pane is automatically hidden For 2 way tube sorting The following items are displayed when 2 Way Tubes is selected in Sorting Method Sort Control pane Configures and controls droplet sorting Sort Start Sort Stop Starts stops sorting Load Collection Unload Collection Loads unloads the collection stage Sorting Method Selects the sorting method Set the sorting method to match the collection devices loaded on the collection stage The sorting parameters vary depending on the selected sorting method Mode Selects the sorting mode Cell Size Displays the current cell size setting Sorting mode e Ultra Purity e Purity e Semi Purity e Normal e Semi Yield e Yield e Ultra Yield e Single Cell Cell size e Regular Cell e Large Cell For details about each sorting mode see Sorting Mode page 93 Main Window e When a Large cell option is selected the side stream deflection angle is adjusted for sorting of cells of larger size e The Large cell options are not displayed if sorting calibration is not performed Select a Large cell option if droplet formation is irregular or the droplet stream is hitting the side wall of the collection tubes or is missing the tubes
284. sed 1f the level in the DI water tank drops too low Filling the DI Water Tank Filling the Sheath Fluid Tank Filling the Sheath Fluid Tank Fill the sheath fluid tank on the fluidics cart with sheath fluid Sheath fluid is used as a transport media to deliver sample fluid to the sorting chip and to clean the sample line Before starting and shutting down the SH800 make sure that there is sufficient sheath fluid in the sheath tank and refill as required Sheath tank For details about refilling the sheath tank on the fluidics cart see Refilling the Sheath Tank page 146 Always place the sheath tank in the center of the tray area The fluid level detector may not operate correctly if the tank is placed off center Tip An alarm is raised if the level in the sheath tank drops too low Filling the Ethanol Tank Fill the ethanol tank on the fluidics cart with ethanol Ethanol is used during ethanol cleaning to disinfect the fluidics system Always check the level in the sheath tank and refill as required before running ethanol cleaning rT Coo EN FS TD A Ethanol tank For details about refilling the ethanol tank on the fluidics cart see Cleaning the Internal Sheath Line and DI Water Line using Ethanol page 188 Always place the ethanol tank in the center of the tray area The fluid level detector may not operate correctly if the tank is placed off center Tip An alarm is raised 1f
285. ser and displays the login window page 42 Help Displays the Help window for accessing the Operator s Guide and to check software version information Sorting control pane Configures and controls droplet sorting It also displays the current status of sorting fluid tank levels lights and laser sources The displayed items vary depending on the model and the sorting method The sorting control pane is a collapsible window element The panel can be shown or hidden as required Tip When the unit is calibrated in analyzer mode the sorting control pane is automatically hidden Name and Function of Parts Experiment Acquisition control area Used to configure and control data acquisition and work with experiments Acquisition tab Displays the controls used for acquiring and recording data Experiments tab Displays experiments and the components in an experiment used to acquire and analyze data in a hierarchical list view File window Example Information tab Change Password if Current password re Institution Information ution information Editable by administrator only Go User Preference Change software default setting Menu items Displays the following menu items Information Displays commands used to configure user passwords and institution information and to add manage user accounts New Creates a new experiment based on saved templates and or recent experiments
286. set a threshold level for the pulse height for one of the channels BSC FSC FL1 to FL6 to discriminate between real events and background noise The threshold level for a channel sets the minimum light detector output that is required to record an event When the trigger is set data is acquired only for events for which the detector output exceeds the threshold level Values below the threshold level are ignored The FSC channel threshold level is the most commonly used trigger as this channel is less susceptible to the effects of background noise The pulse at the threshold level can also be effectively widened to increase the region of the pulse included in the detected signal This is achieved by adding pulse data that was digitized just before the pulse rises above the threshold value and just after the pulse falls below the threshold value These two additional components are called forward window extension FWE and back window extension BWE respectively Window extensions are used to provide more accurate measurements of the area pulse parameter When the window extensions are set to zero only the area above the threshold level is included in the area calculation By setting window extensions before and after the threshold crossing points a more accurate determination of the total area under the pulse can be achieved Intensity Threshold Time The optimum length of the window extensions depends on the rise height a
287. sheath fluid in the sorting chip An agitation unit is provided to maintain consistency of the sample fluid during testing The unit 1s switched on off from SH800 Software Fluid Flow Sheath Fluid Sheath fluid is supplied from the sheath tank in the fluidics cart A level sensor in the fluidics cart monitors the level in the tank The sensor output is displayed on the LCD monitor and on the bottom right of the main window in SH800 Software A warning is displayed if the level gets close to empty The tank is pressurized using regulated compressed air which is used to force the sheath fluid through the fluidics system components at constant pressure avoiding the pressure fluctuation problems common to pumps When Start 1s clicked in SH800 Software to begin sorting sheath fluid is forced under pressure through the sheath filter and into the sheath fluid inlet port on the front of the sorting chip A charging electrode mounted near the inlet port is used to electrostatically charge the sheath fluid for sorting The sheath fluid flow rate is controlled by the Sheath Pressure setting in SH800 Software The sheath pressure determines the speed of the sheath fluid flow through the sorting chip A low flow rate exposes the cells in the sample fluid to a longer duration of laser excitation energy with larger gaps between cells providing the highest fluorescence signal If using fluorochromes with low efficiency luminescence decreas
288. splaying the Compensation Settings dialog see To edit the spillover matrix directly page 82 e Adjusting the position of events shown on a plot also adjusts the position of all events on other plots Accordingly you should add plots for all fluorescence parameters to the worksheet before adjusting compensation manually in order to monitor the affect of adjusting the compensation on each plot To adjust compensation graphically on plots You can adjust fluorescence compensation graphically on the worksheet using a mouse 1 Display the worksheet for the tube whose compensation you want to modify 2 Enable Apply Compensation on the Compensation tab of the ribbon 3 Click Manual Compensation on the Compensation tab of the ribbon to activate manual compensation mode A diagonal line appears on plots that have fluorescence parameters on the X and Y axes CD45 10 105 104 CD3 PE A Compensated 102 CD56 Brilliant Violet 421 4 Place the mouse cursor over the plot and drag the plot to change compensation for the target parameter The mouse cursor changes to a horizontal double ended arrow cursor in the left half of the plot and a vertical cursor in the right half of the plot The displayed events move on the plot to reflect the changed compensation CD45 108 104 CD3 PE A Compensated 2 102 10 10 103 104 104 108 CD56 Brilliant Violet 421 D When finished c
289. ste catcher page 157 and clean separately Check the drip tray in the main unit and fluidics cart for leaks and spills regularly Cleaning the splash guard 1 Moisten a soft lint free laboratory use cloth with water and wipe the splash guard Take care to clean right into the edges and corners 2 Spray a clean soft lint free cloth with ethanol 70 from a spray bottle and wipe the guard 3 Moisten a clean soft lint free cloth with water and wipe the guard Cleaning the Deflection Plates The deflection plates in the sorting area should be removed and cleaned when cleaning the sorting area to ensure the area is not contaminated between sorts With SH800 Software shut down turn off the power supply using the MAIN POWER switch on the rear panel 2 Open the sorting area door on the front panel 3 Pull open the transparent safety cover to access the deflection plates The cover is held closed by a magnetic latch Cleaning the Deflection Plates soueUusUle g Jaydeyy 155 soueUslUle g Ja deuy 156 4 Remove the two plastic bolts securing each deflection plate by hand and remove the deflection plates 5 Clean the deflection plate mounting plate using a lint free laboratory use cloth moistened with water Cleaning the Deflection Plates 6 Clean the deflection plate The electrode surfaces of the deflection plates have been treated with a water repellent coating Wip
290. stored in the database and has no effect on the available database capacity Import Experiment Data dialog The Import Experiment Data dialog is displayed by clicking Import in the Database window The Import Experiment Data dialog is used to import data created on another SH800 into this instrument Import Experiment Data Elapsed Time 00 00 00 Import Data Specifies the experiment data to import Click Browse to specify a file to import Progress bar Displays the import progress Start Starts importing the specified data Close Closes the dialog Export Experiment Data dialog The Export Experiment Data dialog is displayed by clicking Export in the Database window The Export Experiment Data dialog is used to export data created on this unit 0 98 008 000090 Selected Experiment List E Experiment 2 18 2014 5 59 14 PM EJ Experiment 2 23 2014 3 14 37 PM Memo D D User Selects the owner of the experiments to export Type Selects whether to export experiments or experiment templates Search fields Searches for experiments by date and keyword Experiment list Displays a list of experiments that satisfy the specified search criteria Select the experiment s you want to export Selecting an experiment in the upper list displays information about the experiment in the lower list O gt gt All gt gt Clicking gt gt adds the e
291. support guide If the error reoccurs please contact your service representative Unload Sample tube Error Failed to move the sample stage If the error reoccurs please contact your service representative Load Sample tube Error Please reload the tube If the error reoccurs please contact your service representative Load Sample tube Error Failed to move the waste tray Please reload the tube If the error reoccurs please contact your service representative Load Sample tube Error Failed to lift up the sample loader Please reload the tube If the error reoccurs please contact your service representative Error Messages Shut down the cytometer and turn off the compressed air supply Pull the sample line connector out slightly from the chip loader and turn the Emergency thumbwheel to eject the chip Insert a new chip and try to load the chip again If the problem persists contact your Sony distributor for service Check the connection between the cytometer and the host computer and reboot the SH800 Software If the problem persists contact your Sony distributor for service Check that the compressed air supply is supplying rated pressure Shut down the cytometer and turn off the compressed air supply Clean or replace the sample loader O ring If the problem persists contact your Sony distributor for service GUI Error Message Recommended Solution Load Sample tube Error Failed to
292. t Close Closes the dialog Vacuum Fan Control dialog The Vacuum Fan Control dialog is displayed by clicking Vacuum Fan in the Safety group on the Cytometer tab of the ribbon The Vacuum Fan Control dialog is used to set a timer for operation of the vacuum fan The fan is used to extract aerosols microparticles of biological and other matter in suspension created by sorting from the sorting area Vacuum Fan Control Vacuum duration 00 01 00 v Elapsed Time 00 00 00 00 01 00 Start Vacuum duration Set the duration for fan operation Setting the duration to 00 00 00 allows you to operate the fan for an unlimited duration When you want to stop the fan in such cases click Stop Elapsed Time Displays the elapsed time since starting the fan Start Starts the fan Stop Stops the fan Close Closes the dialog Cytometer Settings dialog The Cytometer Status dialog is displayed by clicking Settings in the Settings group on the Cytometer tab of the ribbon The Cytometer Settings dialog is used to configure instrument settings adjust sort parameters and other functions e Control tab This tab is used to configure instrument settings Cytometer Settings Control Chip Information Cytometer Information Maintenance Laser Threshold 405mm On Turn Off Channel FSC 7 j es R p ar 7 a 488nm On Turn Off Value 5 00 m Wi 561nm On Turn Off Sensor Gain FSC 4
293. t have a selected detection channel pulse parameter are included in the compensation panel for the sample group in the experiment structure Creating a Customized Experiment e The fluorochrome and marker names are optional but recommended e Check that the fluorochrome and marker names assigned to detection channels correspond with the laser configuration and optical filter pattern For details see Fluorochrome Detection Matrix page 204 5 Modify the structure of the sample group as required For details see To add a sample tube page 72 and To delete a sample group or tube page 73 6 When finished click Add Sample Group in the bottom right hand corner of the window The sample group is added to the experiment To add a sample tube Use the following procedure to add a sample tube to the selected sample group 1 Click Add Tube in the Create Experiment window Add Sample Group Add Tube The Add Tube dialog appears 2 Select a sample tube template or recent sample tube in Tube Templates Tube Templates Public Templates Blank Tempiate My Templates Recent Tubes Top 10 v Tube 2 Tube 1 Tube i Tube 5 Tube 4 Tube 3 Tube 2 Tube 1 Tube 1 Tube 8 3 Enter a name for the sample tube and modify other settings as required The name of the sample tube is a required item Tube Information Name Tube 7 4 When finished click Add Tube in the bot
294. t in serious injury or electrical shock Do not touch the deflection plates while power is connected to the main unit The sorting area is sealed during operation to prevent contamination of the air by aerosols generated by falling droplets and to prevent air circulation in the room affecting the accuracy of the droplet sorting Negative OT Satellite droplet u amplitude and frequency of vibration of the sorting chip to control the position of the breakoff point and the timing of the electrostatic charging pulse Manual droplet control is also supported Last attached droplet Satellite droplet The side stream monitor detects the position of the side streams The output image of the side stream camera is displayed in the bottom of the main window in SH800 Software The output image is also used by control circuits to automatically adjust the angle of deflection of the side streams so that they are directed into the collection tubes The collection stage supports 15 ml conical tubes and 5 ml round tubes inserted in collection tube holders Right side stream pressure vacuum suction helps reduce the risks associated with aerosol generation Waste fluid may contain biological chemical or other agents Always wear gloves and other protective clothing mask and goggles as required when handling samples The handling of waste fluid should be performed in accordance with biological hazard handling safety procedures
295. t to leave the chip inserted the chip must be realigned To align the chip click Chip Alignment on the Cytometer tab of the ribbon When finished turn on the compressed air supply 8 Close the flip up door Ejecting the Sorting Chip Manually 185 soueuajUleW g sajdeyuy 186 9 Turn on the main power supply Cleaning and Handling of Optical Filters Cleaning and Handling of Optical Filters The bandpass optical filters and longpass optical filters in the detection module require only light cleaning to remove any dust on the surface of the filters The filters should be checked and cleaned every 6 to 12 months or more often if the optical filter pattern is routinely changed The optical filters are high precision components that require the utmost care If an optical filter is scratched or damaged in any way it must be replaced Remove an optical filter by placing your finger under the lip of the retaining holder and pulling directly out 2 Grip the sides of the optical filter and withdraw it from the filter slot Do not touch the surfaces of the optical filters It is recommended that only one optical filter be withdrawn at any one time to ensure that it is returned to the original slot 3 Remove loose dust or particles using a canned air blower or a bulb puffer hand duster similar to those used in photography Direct the airflow across the surface at an oblique angle If necessary ge
296. t use a 96 well plate holder e Always use a 96 well PCR plate holder when using 96 well PCR plates Do not use a 384 well plate holder Loading a 384 well PCR Plate Place the 384 well PCR adapter in the 384 well plate holder and place a 384 well PCR plate on the adapter Place the 384 well PCR plate into the adapter with the A1 well at the front left of the instrument 384 well PCR plate 384 well PCR adapter 384 well plate holder Always use a 384 well PCR plate holder and 384 well PCR adapter when using 384 well PCR plates Do not use a 96 well PCR plate holder Removing the Plate Holder Adapter SH800ZP only When running 2 way tube sorting the plate holder adapter must first be removed Remove the screw securing the adapter using a coin or screwdriver and remove the adapter Plate holder adapter Preparing Collection Tubes 37 uoneledaig zieideyo HN uo eied ld guiajdeuy Configuring Users This section describes how to manage user accounts in SH800 Software For details about starting the SHSOOO instrument and SH800 Software see System Startup page 41 and Logging In page 42 Adding Users Only administrators can add new users 1 Click the File tab then click Information in the menu on the left 2 Click Account Settings amp Change Password Change your current password re Institution Information Show your j stitution information Edi
297. t will vary depending on the axis type log linear or biexponential e The negative and positive populations may or may not appear as diverse populations on 2D plots depending on the type of cells in the sample and the selected fluorochromes For example if samples contain cells that are actively acquiring or losing the antigen with the fluorochrome marker the two populations may tend to merge into one another e SH800 Software also supports manual specification of the fluorescent compensation parameters See Adjusting Fluorescence Compensation Manually page 81 Miscellaneous Series Lineup The SH800 cell sorter is available in six different models equipped with a combination of one two three or four excitation lasers of different wavelengths The models are indicated by a two letter suffix AC to FC SH800 Model No of asers Laser wavelengths om _ LE SH800DC 488 561 LE SH800EC 488 405 638 LE SH800FC 488 405 638 561 SH800Z Modei No ot lasers Laser wavelengths om SH800ZP Laser wavelengths nm 406688 LE SH800ZDP 488 638 405 488 488 561 LE SH800ZFP LE SH800ZGP LE SH800ZHP e 488 nm blue laser standard e 405 nm violet laser 405 488 638 561 405 488 561 488 638 561 Appendix e 561 nm yellow green laser e 638 nm red laser The fluorochromes that can be excited and the resulting emission spectra that can be detected by each laser configuration
298. tabase 1 Click Database on the File tab of the ribbon then click Import The Import Experiment Data dialog appears Import Experiment Data 2 Click Browse to specify the folder where the experiment data you want to import expdat file is stored 3 Click Start The import status is displayed in the progress bar A confirmation dialog appears when importing is completed 4 Click OK 5 Click Close to close the dialog Sharing Experiments Sharing Experiments You can share experiments with other users Shared experiments allow multiple users to work with the experiment data on worksheets To share an experiment Select the experiment you want to share in the Experiment Explorer and click Send to Public on the Experiment tab of the ribbon Alternatively you can right click an experiment and select Send to Public from the context menu public user E Experiment 777 The shared experiment is duplicated in the public node in the Experiment Explorer Tip Other users can access the shared experiment under the public node After an experiment is shared the shared experiment and the original private experiment exist as independent experiments with no interaction between the two Searching Experiments You can search experiments by date and keyword Enter the search criteria in the Experiment Explorer of the Experiments tab on the left side of the main
299. table by administrator only The Account Settings dialog appears 3 Click Add Account Settings Faas Account Properties userl Username administrator Full Name Organization Edit Change Password Close The Add Account dialog appears 38 Configuring Users 4 Set each field then click OK Add Account Username Password Confirm Password Full Name Organization Accessibility Pa Cancel lo Username Enter the user name of up to 32 alphanumeric characters used when logging in to SH800 Software required item Password Enter the password comprising up to 20 alphanumeric characters used when logging in to SH800 Software required item Confirm Password Enter the same password again for confirmation required item Full Name Enter the full name of the user Organization Enter the name of the organization Accessibility Place a check mark in the checkbox if you want to temporarily disable a general user account The user is registered in the user list in the Account Settings dialog User list Account Settings Account Properties Username administrator Full Name Organization Edit Change Password Close Editing User Settings Only administrators can edit user settings Select the user you want to edit in the Account Settings dialog then click Edit Selecting a user in the list displays the settings for th
300. table for most cell sorting applications Cells ranging from approximately 1 um to 20 um in diameter can be analyzed and sorted The size of the sorting chip nozzle affects the droplet size the gap between droplets and the consumption of sample fluid and sheath fluid Sample fluid can be loaded in 0 5 ml micro tubes 1 5 ml micro tubes 5 ml round tubes or 15 ml conical tubes for use with the SH800 Each type of tube fits into a corresponding tube holder for the sample tube The tube holder all fit into a D cut slot in the base of the sample loader After the sample is loaded and Start 1s clicked in SH800 Software the sample door closes and the sample tube containing the sample is raised into the injection chamber The chamber is hermetically sealed and then pressurized using regulated compressed air The pressure inside the injection chamber forces the sample fluid up through the sample probe and into the sample line connected to the probe at the top of the chamber Sample fluid flows through the sample line and into the sample fluid inlet port on the front of the sorting chip via a pinch valve which opens closes the sample line to control the sample flow The sample fluid flow rate is controlled by the Sample Pressure setting on the main window in SH800 Software The sample pressure determines the speed of the sample flow which indirectly controls the distance between cells in the sample fluid flow as they are injected into the
301. ter Settings DI filter de bubble DI filter de bubble Tip The DI water filter de bubble function should be run at least three times after changing the DI water filter For details see Changing the DI Water Filter page 177 Main Window e Advanced Settings dialog The Advanced Settings dialog 1s used to adjust sort parameters manually Advanced Settings ptions m re Opti ge Z x 10 A 10 z gt Ip v Dro Carr umina ight A Laser 2048 LED 6 Vv 1000 age A lt 4 gt Close Droplet Control Contains adjustments related to droplet formation Drop Drive The drop drive setting adjusts the amplitude of the piezoelectric element drive voltage The frequency sets the cycle time of vibration of the sorting chip The vibration of the sorting chip causes the stream to break into droplets after the stream is emitted from the chip nozzle The drop drive is optimized during auto calibration Drop Clock The drop clock setting adjusts the frequency of the vibration of the piezoelectric element drive voltage The frequency sets the cycle time of vibration of the sorting chip The drop drive is optimized during auto calibration Sort Delay The sort delay setting adjusts the number of vibrations applied to the sorting chip as cells pass between the optical detection point and the breakoff point It is used to determine the timing of the electrostatic charging pulse applied to the sheath
302. ter from the DI water tank when cleaning the probe If a check mark is placed in this checkbox you should load a sample tube containing DI water after cleaning the sample probe to clean the outside of the probe Area Light Control Sample tube area light automatic control Specifies whether the sample loader light turns on automatically loading unloading a sample e Off e On Collection area light automatic control Specifies whether the collection area light turns on automatically when loading unloading collection tubes Experiment tab Defines default settings related to experiments User Preference Sort Define default settings for experiment Reset Cytometer Sre EEEE New Experiment Set default action after creating new experiment Don t ask about action after creating new experiment Compensation Default Action User Defined Color Assign First Tube M Start Co FCS File Explorer Set default setting of public user s data display Expand Public User s data On Off Export Experiments Set default export folder of exported files Default Export Folder New Experiment Don t ask about action after creating new experiment Sets whether to display a dialog prompting you to select the next action after creating an experiment using the Blank template Default Action Selects the default action to take after creating an experiment e Assign First Tube Assigns the first tube in prep
303. tes 8 well PCR strips 96 well PCR plates 96 well PCR plate holder Name and Function of Parts 19 roneo va WIN MAPIAJOAQ Ja deuyD 20 Collection tubes multi well plates 384 well plates 384 well PCR plates 384 well PCR plates 384 well plate holder 384 well PCR adapter 1 Options For details about loading tubes and the types of collection tubes see Preparing Collection Tubes page 33 Sample loader area Injection chamber Sample loader Holder Holds the collection tube device Collection stage Loads the collection tube device holder Plate holder adapter multi well plates Used for loading multi well plate collection devices When running 2 way tube sorting remove the adapter and load the corresponding collection tube holder e Always wear laboratory use gloves mask protective goggles and other protective clothing when handling sample fluid to provide protection against biohazards e Clean the fluidics system regularly using a sodium hypochlorite liquid solution bleach to prevent clogging of the sample line sample probe and suction pump e Load the sample tube with its cap removed Name and Function of Parts Sample tube Sample tube holder Agitation unit Injection chamber Extracts sample fluid from the sample tube through the sample probe and forces the fluid into the sorting chip Sample loader Loads a
304. tes on plots but by specifying an equation for the gate The populations defined by Boolean gates can be displayed on plots just like populations from physical gates All statistics functions available for physical gates are also available for Boolean gates Physical gates and Boolean gates can be selected as the sorting criteria when sorting For details see Sorting page 93 Adding a Physical Gate You add a physical gate directly to plots 1 Select a plot on which to add a gate Change the plot zoom as required in the Worksheet View group on the Worksheet Tools tab of the ribbon to make it easier to draw the gate t Violet 4 400 200 150 100 50 Adding Gates 2 Click the desired gate type button in the Gate group on the Plot Tools tab of the ribbon and draw a gate on the plot Alternatively right click the plot then select Create gate and the desired shape of gate from the context menu Rectangle Adds a rectangle gate Ellipse Adds an ellipse gate Polygon Adds a polygon gate Quadrant Adds a quadrant gate A quadrant gate is created on a plot by clicking the mouse at the intersection point of the four quadrants The four quadrants lt gate_name gt Q1 to lt gate_name gt Q4 are added to the plot Drag the intersection point to change the size of the quadrants Linear Adds a linear gate Linear gates can be added to histogram plots only The g
305. th tank If air is leaking reattach the lid until it is correctly sealed Check that there are no air bubbles in the sheath filter If the problem persists there may be air bubbles trapped in the sheath filter Remove the air bubbles from the sheath filter See Releasing Air in the Sheath Filter and Sorting Chip page 181 If the problem persists there may be salt deposits in the flow channels Log off and restart in maintenance mode Running Maintenance Mode page 158 The sorting chip is ejected automatically Check that the surface of the chip is not dirty or smudged then perform chip realignment If the sorting chip is dirty or smudged replace the chip If the problem persists contact your Sony distributor for service Shape of droplets is irregular The sorting chip may contain trapped air Check that there is no air trapped in the sheath filter Click Chip in the De bubble group on the Cytometer tab of the ribbon several times as required to clear the chip Check that there are no air bubbles in the sheath filter If the problem persists there may be air bubbles trapped in the sheath filter Remove the air bubbles from the sheath filter See Releasing Air in the Sheath Filter and Sorting Chip page 181 lf the problem persists there may be salt deposits in the flow channels Log off and restart in maintenance mode Running Maintenance Mode page 158 The sorting chip is ejected automatically Check
306. the event is sorted in the left side stream left side stream always has precedence Sorting onto 8 well Strips SH800 and SH800Z Check that data acquisition and recording are stopped or paused If data acquisition is in progress click the Stop to stop data acquisition recording or Pause Pause to pause data acquisition recording Sample Group 1 O Tube 1 Status Pause Elapsed Time 00 00 12 Total Event 310 Event Rate 0 eps TE pet Start Record Stop Sample Stop Condition None v Place the 8 well strips or glass slides onto the corresponding holder For details see Preparing Collection Tubes page 33 Open the sorting area door place the holder on the collection stage and close the door 8 well strips holder Slide glass holder e Do not sort sample fluid above the permitted volume into the wells and do not overfill the wells with buffer solution to prevent sample fluid overflow from the wells Sorting 55 uo eiado oiseg Jaldeyuy Kii uoleisdo olseg sa deyuyD 56 e Take care not to spill samples when removing the 8 well strips or glass slides from the multi well plate holder e Ensure that the 8 well strips or slide glass selected in Sorting Method is loaded correctly e Do not open the sorting area door during sorting e Do not attempt to remove the 8 well strips or slide glass whil
307. the level in the ethanol tank drops too low Preparing a Sample Loading a Sample Tube in a Sample Tube Holder Insert the sample fluid for analysis or sorting in a sample tube and place the tube in the corresponding sample tube holder The SH800 supports the following sample tubes Each sample tube is used with a corresponding holder that mounts in the sample loader 0 5 ml micro tubes Sample tube holder 0 5 ml 1 5 ml micro tubes Sample tube holder 1 5 ml 5 ml round tubes Sample tube holder 5 ml 15 ml conical tubes Sample tube holder 15 ml Load the sample tube into the corresponding size sample tube holder Always remove the sample tube caps when performing measurements 0 IT O na 5ml round tube 0 5 ml micro tube 1 5 ml micro tube 15 ml conical tube The tube holders for micro tubes have a recess at the top to hold the cap clear of the tube opening Filling the Ethanol Tank Preparing a Sample 31 uoneiedaig guaideyy HIN uonweiedald gzudajdeyug 32 Recess Always wear laboratory use gloves mask protective goggles and other protective clothing when handling sample fluid to provide protection against biohazards Also take care when loading the sample tube in the tube holder to avoid sample fluid spills and breakage of the sample tube Before using a tube check that there is no dust or other foreign matter in the tube Preparing Automatic Setup Beads Pr
308. the plot view has stabilized 14 Check the position of the gate on the density plot and the gate for the positive population on the histogram adjust the gate if necessary then click Next All Events 1 000 300 i SRPNA seai Lads 3 29 51 72 93 30 aot 10 108 104 10 N FSC A FITC A Compensated 15 Repeat steps 11 to 14 to record each of the control tubes in sequence Performing Fluorescence Compensation 63 uoneiedo oiseg g13 deyo HIN uoleisdo olseg Jsa deuyD 16 After recording all control tubes click Calculate Matrix on the Compensation tab of the ribbon Compensation Wizard Vv 1 Gain Settings v 2 Acquiring Sample Data v Brilliant Violet 421 FITC PE APC PE Cy7 3 Calculation The Calculate Compensation Settings dialog appears 17 Check the target compensation panel then click Calculate Calculate Compensation Settings Spillover matrix will be calculated from compensation tubes data in compensation pane Target Compensation Panel Spillover Matrix userl Expenment 12 30 2013 10 26 54 PM Sample Group 1 Compensation Panel Compensation Tube Gate x amp Negative control HH Briliant Violet421 je 5 bs Es 5 v fc is Brilliant Violet 421 100 00 000 0 00 000 ooo 000 PAD His FITC 0 00 100 00 0 00 000 000 0 00 Bc His 5 PE 0 00 0 00 100 00 0 00 000 0 00 amp Percr cyss His B APC 0 00 000 0 00 100 00 0 00 0 00 Breg7 ws 2 PercP cys5 000 0 00 0 00 0 00
309. the selected experiment Rename Renames the selected experiment Compensation Panel The following menu commands are displayed when right clicking a compensation panel icon in the Experiment Explorer Compensation Wizard Displays the Compensation Wizard page 61 for recording control tubes in a compensation panel Show Matrix Displays the Compensation Settings dialog page 124 for viewing the spillover matrix and loading saving the spillover matrix Calculate Matrix Displays the Calculate Compensation Settings dialog page 125 for calculating the coefficients of the fluorescence compensation spillover matrix based on the negative control tubes and the compensation panel Export FCS File Displays the FCS File Export dialog page 117 for exporting the tubes in the selected compensation panel in FCS 3 0 or FCS 3 1 format Control tubes in a compensation panel The following menu commands are displayed when right clicking a control tube in a compensation panel icon in the Experiment Explorer Assign Assigns the selected control tube as the active tube for data acquisition and recording Open Worksheet Opens the worksheet tab for the selected control tube Export FCS File Displays the FCS File Export dialog page 117 for exporting the selected control tube in FCS 3 0 or FCS 3 1 format Reset Data Deletes the data recorded for the control tube Sample groups The following menu commands
310. ther information for the chip appears 2 Check that the displayed information is correct then click Next Initial Instrument Setup Vv 1 Chip Detection Chip Details If the information is correct click the Next button 2 Chip Information Detected Information umber J82aR1N 100um Instrument Settings Sheath Pressure 20PSI Target Event Rate 5 000eps The currently loaded sorting chip if one is present is ejected from the chip loader for removal Tip If you wish to load a different chip click Back to return to the previous screen then scan the QR code for the different chip Initial Instrument Setup Please exchange the chip Vv 1 Chip Detection vV 2 Chip Information 3 Chip Loading Open the flip up door to access the chip loader Remove the current sorting chip if one is present and insert the new chip into the chip slot at the top of the chip loader EMERGENCY LOAD UNLOAD Insert the chip into the slot with the Sony logo and nozzle size mark at the top facing toward you Place the previous chip in its original packaging The chip and its corresponding QR code should be kept together at all times so that they are not mixed up with other chips e An error message appears if the sorting chip is inserted in the incorrect orientation e Do not insert any objects other than Sony sorting and cleaning chips designed for the SH800 into the chip insertion slot e The
311. tics Editor dialog page 92 to configure the statistics displayed in the Gates and Statistics table Favorite Settings gt Save as Favorite Saves the current worksheet settings as the favorite default worksheet settings Restore Favorite Restores the saved favorite worksheet settings as the current worksheet settings Copy Worksheet Picture Copies the selected plot or Gates and Statistics table to the clipboard for pasting in other software for example to produce reports Print Displays the Print window page 108 for printing the analysis results displayed on the worksheet Custom Print Displays the Custom Print dialog page 128 for selecting the items to print Properties Displays the Property Window dialog page 130 Plots The following menu commands are displayed when right clicking a plot Refresh Updates the information displayed on the worksheet Auto Adjust Automatically adjusts the ranges of the X axis and Y axis of all plots to their minimum and maximum measured values Remove Removes the selected plot Duplicate Duplicates the selected plot Copy All Regions Paste Region Copies all gates on a plot and pastes them onto another plot Create Gate gt Rectangle Creates a rectangular gate on a dot plot or density plot Ellipse Creates an elliptical gate on a dot plot or density plot Polygon Creates a polygonal gate on a dot plot or density p
312. ting Load Collection Unload Collection Loads unloads the collection stage Sorting Method Selects the sorting method Set the sorting method to match the collection devices loaded on the collection stage The sorting parameters vary depending on the selected sorting method Sort Settings Specifies the default sorting configuration Clicking this button displays the Sort Settings dialog For details about the Sort Settings dialog see Sorting page 53 and Adjusting the Sort Position SHSOOZP only page 99 Sort Statistics pane Displays the statistics about events during sorting Total Elapsed Time Displays the cumulative elapsed time since the start of sorting of the whole plate Total Progress Displays the ratio of sorted wells to the total number of wells Sort ID Displays the ID of the well currently being sorted Well Number Displays the number of the well currently being sorted Sort Mode Displays the sorting mode of the well currently being sorted Cell Size Displays the current cell size setting Sort Gate Displays the sort gate of the well currently being sorted Stop Count Displays the number of events for the automatic stop condition of the well currently being sorted Plate image Displays the layout of the wells Double clicking the plate image or right clicking and selecting Show Sorting Report from the context menu displays the plate image i
313. tion see Cleaning the Sample Fluidics System using Bleach page 152 When sample line cleaning finishes the Fluidics Check screen appears When the fluidics check is completed the Auto Calibration screen appears Cleaning the Sample Line 45 uoneiedo oiseg g13 deyo Kii uoleisdo olseg Jsa deuyD Automatic Calibration After configuring the excitation lasers and emission detection optical filters the startup procedure automatically performs sorting chip alignment droplet calibration sort delay calibration and side stream calibration using automatic setup beads on the Auto Calibration screen Auto Calibration Please set calibration beads and click OK button O Analyzer mode Calibrate the chip alignment only Skip Auto Calibration e You can click Skip Auto Calibration to bypass automatic calibration and proceed directly to the SH800 Software main window Bypassing automatic calibration is useful for quickly accessing the main window to perform analysis of existing data Do not bypass automatic calibration if you plan to run any new samples e You can align the sorting chip and calibrate the sorting system later using Chip Alignment and Sort Calibration respectively on the Cytometer tab of the ribbon from the main window Insert 1 ml or more of undiluted automatic setup beads in a sample tube and place the tube in the corresponding sample tube holder For deta
314. to begin ethanol cleaning Follow the on screen instructions Ethanol Cleaning Click Start to initiate Ethanol cleaning procedure Warning The Ethanol Cleaning procedure cannot be aborted once the sheath filter has been bypassed For details about ethanol cleaning see Cleaning the Internal Sheath Line and DI Water Line using Ethanol page 188 When ethanol cleaning finishes the login screen reappears Running Waste Line A Maintenance The instrument should be started in maintenance mode to prevent the buildup of salts and clean waste lines Waste Line A 1 Click Maintenance on the bottom right of the SH800 Software login screen Cell Sorter SH800 semam Panor The Maintenance wizard appears 2 Select WasteA maintenance then click Start 7 Maintenance Please select maintenance mode Ethanol cleaning Internal sheath line and DI water line are cleaned with ethanol WasteA maintenance O It is recommended that if you have not used for a long time to perform WasteA maintenance Sample line exchange Exchange sample line and probe for new one The WasteA maintenance wizard appears 3 Click Start to begin WasteA maintenance Follow the on screen instructions In WasteA maintenance the fluidics system is cleaned using a sodium hypochlorite solution bleach cleaning the DI water lines are cleaned and the internal sheath lines are cleaned in that order W
315. tom right hand corner of the window To delete a sample group or tube Select the sample group or tube you want to delete in the structure tree in the Create Experiment window and click Delete Selected Item PE APC PerCP Cy5 5 amp W PE Cy7 lt Add Tube Delete Selected Item Add Sample Group Configuring an Experiment This describes how to configure each component in an experiment 1 Expand the target experiment in the Experiment Explorer and double click Measurement Settings in the sample group you want to configure A public 0 A usert 24 4 F Experiment 12 26 2013 4 26 01 PM Experiment Information 4 E Sample Group 1 amp Sample Group Information Q Compensation Settings a Compensation Panel 4 Tube 1 1 Tube Information The Measurement Settings dialog appears 2 Set each item Sample Group 1 Measurement Settings Parameter Settings Acquisition Select Fluorochrome Area Height Width A y Brilliant Violet 421 FITC PE APC F this SampleGroup has a recorded tube you cannot change Instrument Settings Laser Threshold Sensor Gain a4 Channel FS FSC 4 W 488mm On Value 5 00 FLI 40 0 FL3 40 0 FL5 Sample Pressure 4 AD Advanced Settings Forward Window Extension Back Window Extension OK Cancel These parameters are configured in the Detector amp Threshold Settings dialog page 79 Select the p
316. ton for controlling agitation of the sample fluid Agitate Turns the sample loader agitation unit on off Safety group Contains a tool button for running a suction fan for safety ae Vacuum Fan Safety Vacuum Fan Displays the Vacuum Fan Control dialog page 121 for setting a timer for operation of the vacuum fan The fan is used to extract aerosols microparticles of biological and other matter in suspension created by sorting from the sorting area Setting the duration to 0 runs the fan without the timer function Exchange group Contains a tool button for exchanging the sorting chip CF Sh chio Sampie Line Chip Ejects the sorting chip and displays the Chip Exchange wizard for inserting a different sorting chip For details see Exchanging the Sorting Chip page 67 Sample Line Displays the Sample Line Exchange wizard for changing the sample line For details see Changing the PEEK Sample Line PEEK sample line compatible models page 169 or Changing the Sample Line and Probe Non PEEK sample line compatible models page 172 Tip The sorting chip must be replaced or reinserted after changing the sample line Settings group Contains tools button for configuring advanced settings related to the instrument ale iia Settings Droplet Senna Settings Displays the Cytometer Settings dialog page 121 for configuring the instru
317. trument settings as a previous experiment enables direct comparisons to be made between the results obtained even if the experiments were conducted at different times You can also configure many of the same parameters using the Compensation Wizard and on the Control tab of the Cytometer Settings dialog For details see Performing Fluorescence Compensation page 61 and Cytometer Settings dialog page 121 1 Click Detector amp Threshold Settings in the Acquisition tab of the Experiment Acquisition control pane Status Ready Elapsed Time 00 00 00 Total Event 0 Event Rate a Ee B Sample Stop Conditior None v Detector amp Threshold Settings Sample Pressure 4 v The Detector amp Threshold Settings dialog appears 2 Set each item Detector amp Threshold Settings Threshold Channel FSC 5 0 alue Sensor Ga n F L A a La i B L a FL1 AO v L aaa 40 E T FL2 aaa aa 0 AD FL3 PE 0 40 T FL4 APC 00 EG FL5 PerCP Cy5 5 40 0 An no r r FLG PE Cy7 40 0 f Threshold Specifies the threshold for event detection Channel Selects the channel FSC BSC FL1 to FL6 used to trigger the detection of events The default trigger channel is FSC Value Sets the threshold value for the trigger channel as a percentage of the output signal of the detector for the trigger channel The default value is 5 00
318. ts can be modified but they cannot be added nor deleted e To save the current compensation settings click Save To load compensation settings for the sample group click Load e To reset the compensation settings click Reset Click Close to close the dialog Analysis Chapter Data is analyzed using various tools available on the worksheet This chapter describes gate settings used for analysis of the acquired data and for sorting generating reports of data analysis and related tasks For details about content displayed on the main window see Main Window page 113 2 Change the layout of plots as required Adding a Plot You can change the position of plots by selecting the target plot and dragging it to a new location You can add plots to the worksheet Click a button for a new plot of the desired plot type to be added in the Plot group on the Worksheet Tools tab or the Plot Tools tab of the ribbon oe Experiment Cytometer Compensation N TE tes i X li 3 A ion Le Fa Ey BA quisition New lew Dot Mew Remove Duplicate EA Analysis Plot Piot Density Plot Histogram The plot is added to the worksheet The new plot created represents the All Events population Droplet Edit the plots add gates and perform other actions on plots as required See Editing Plots page 84 and Adding Gates page 88 Adding a Plot 83
319. ts the voltage applied to each of the deflection plates Pressure Options tab Sets the sample fluid flow controls and the window extension settings Sort Options Stream Options Pressure Options Flow Control 41 gt 41 gt Flow Control Adjusts the sample pressure and sheath pressure Clicking Standby stops the sheath and sample fluid flow Clicking Ready starts sheath flow again in preparation for a sample Pulse Calculation Sets the forward window extension FWE and back window extension BWE settings used to modify the pulse parameters for data acquisition Tip The setting of the forward window extension and back window extension affect the data acquisition results for Area pulse parameters Main Window Compensation Tab ribbon The Compensation tab of the ribbon has the following buttons Compensation group Contains tool buttons related to fluorescence compensation Heel R R Compensation Show Calculate Apply Manua Wizard Matrix Matrix Compensation Compensation npenss Compensation Wizard Displays the Compensation Wizard for recording control tubes in a compensation panel The compensation settings should normally be configured using the Compensation Wizard to record a negative unstained control tube and up to six positive stained control tubes in a compensation panel The Compensation Wizard then automatically calculates the fluorescence compensation spillover
320. tter FSC back scatter BSC and six fluorescent light FL1 to FL6 channels for multi color analysis The optical filters can be changed to tailor the detection channels for specific measurements e The detection module uses components that are susceptible to static electricity which may cause an error in operation Do not touch the detection module during operation to prevent the transfer of static electricity e If you sense a discharge of static electricity from your body near the optical filters during data acquisition it is recommended that acquisition be paused and then resumed This procedure restores normal gain settings even in the event that static electricity affects the operation of the detection module Deflection plates Waste catcher Collection tubes Holder Collection stage SH800ZP model Deflection plates Waste catcher Collection tubes Holder Collection stage Q Plate holder adapter Fluids may contain biological chemical or other agents When handling collection tubes always wear laboratory use gloves mask protective goggles and other protective clothing Deflection plates Deflects electrostatically charged droplets ejected from the nozzle of the sorting chip sorting the droplets into the designated collection tubes Droplets that contain target cells for sorting are charged as they are ejected from the sorting chip nozzle while
321. uator SH800Z and SH800ZP Troubleshooting This section lists common problems that may occur and the actions to take to resolve each problem Check these items before calling for service If the problem still persists after carrying out the recommended actions contact your Sony distributor Possible Cause Recommended Solution The event rate does not rise during data The sample line may be clogged Click Probe Wash in the Cleaning group on acquisition sorting or adjustment the Cytometer tab of the ribbon to clean the sample line If the problem persists change the sample line page 172 Measurement values cannot be acquired The sorting chip may be clogged or contain trapped air Click Chip in the De bubble group on the Cytometer tab of the ribbon several times as required to clear the chip If the problem persists the objective module lens may require cleaning Contact your Sony distributor for service Droplet image in droplet viewer shakes The sorting chip may be clogged or contain trapped air Click Chip in the De up and down or flows in a curved path bubble group on the Cytometer tab of the ribbon several times as required to clear the chip If the problem persists replace the sorting chip The fluid consistency of the sample may be causing fluctuations in the sample flow Click Agitate on the Cytometer tab of the ribbon to turn on sample agitation Check that there is no air leaking from the shea
322. ude and frequency settings to obtain a stable breakoff point After establishing a stable breakoff point click the feedback control checkbox to enable the breakoff feedback control e The manual amplitude and frequency controls are disabled in this mode e When breakoff feedback control is enabled if the breakoff point moves and cannot be recovered within a predetermined time sorting stops and an error message is displayed on the screen Context Menu The context menu displays common operations that can be accessed by right clicking components in the Experiment Explorer or items on the worksheet Experiment Explorer Context Menu This section describes the context menu items displayed for components in the Experiment Explorer Experiments The following menu commands are displayed when right clicking an experiment icon in the Experiment Explorer New Sample Group Creates a blank sample group Copy Paste Copies the experiment and pastes a new experiment into the Experiment Explorer Duplicate Duplicates the selected experiment Save as Template Saves the selected experiment as a private template Export FCS File Displays the FCS File Export dialog page 117 for exporting the tubes in the selected experiment in FCS 3 0 or FCS 3 1 format Send to Public Creates a duplicate of the selected experiment and places the duplicate under the Public node for sharing with other users Delete Deletes
323. ulse parameters Area Height Width for each detection channel The SH800 supports the acquisition of three pulse parameters area height width in the detector for each of the input channels FSC BSC FL1 to FL6 for each tube in a sample Configuring an Experiment 73 sjuewedxy Buunbyu0D p saideuD HIM s u wn dx Buunbyuog p Je deyD 74 group You select the detection channels for which to acquire data by selecting acquisition pulse parameters for those channels Any combination of pulse parameters can be selected for each detector channel The Area Height and Width pulse parameters are selected by default for the FSC detector channel Only the Area pulse parameter is selected by default for all other detector channels The Area pulse parameters for the FSC and BSC detector channels are always enabled Only detector channels with a pulse parameter selection will appear in the compensation panel 2 Specify a marker name and or fluorochrome name for each fluorescence detector channel You can also enter a fluorochrome name directly from the keyboard Marker names and fluorochrome names if specified appear in the axis labels on plots and fluorochrome names appear for control tubes in the compensation panel Forward scatter FSC and back scatter BSC are measured using the incident laser light not fluorescent light Entries for FSC and BSC are not permitted Fluorochrome names and marker names can be
324. unication on MD1 board If the problem persists contact your Sony distributor for Contact your Sony distributor for scheduled maintenance The pump C may need to be repaired The e p regulator for sheath fluid may need to be repaired The e p regulator for sample fluid may need to be repaired The cooling device for the sample area may need to be repaired The cooling device for the collection area may need to be repaired A reduction in laser output power has been detected Contact your Sony distributor for advice Error Messages GUI Error Message Recommended Solution The LD1 laser is approaching its end of life expiration Laser output does not decrease immediately The laser can ae TF continue to be used but should be replaced as soon as The LD2 laser is approaching its end of life expiration possible The LD3 laser is approaching its end of life expiration The LD4 laser is approaching its end of life expiration Failed to record LD1 laser total hours of use Total irradiation time was not recorded correctly The laser is l still operational but you should contact your Sony distributor Failed to record LD2 laser total hours of use ESON CE Failed to record LD3 laser total hours of use Failed to record LD4 laser total hours of use DI Water Tank is almost empty Refill the DI water tank See Refilling the DI Water Tank Refill the DI water tank following the instructions in the page 149 operator s guide
325. uorescent light when excited by lasers at certain wavelengths and low level fluorescent light at other wavelengths The wavelength of the excitation lasers should be selected to match the fluorochromes for the markers Sheath and sample flow Fluorescent light FL Incident laser light Fluorescence occurs when an electron in a molecule is raised to an excited state as the electron absorbs a photon of the incident excitation light The electron loses some of this energy before the molecule emits a different photon and the electron returns to its ground state The emitted photon has less energy than the absorbed photon so the emitted light has a longer wavelength than the incident light and hence a different color The difference between the peak excitation wavelength and peak emitted wavelength is called the Stokes shift Some fluorochromes are available that exhibit a narrow Stokes shift while others exhibit a wide Stokes shift Multicolor fluorescent light analysis can be used to provide a wealth of structural and other functional information about cells Laser Interrogation Light source 405 nm violet laser 4 laser models The excitation laser module supplies the laser light used to interrogate individual cells as they flow past the optical detection point The excitation laser beams are combined into a single collinear beam and coupled to a single mode fiber to the objective lens module The exc
326. using Sample line exchange on the Initial Instrument Setup screen or using Sample line exchange in maintenance mode For details see Logging In page 42 and Running Maintenance Mode page 158 3 Open the flip up door on the front panel m T 4 Remove the sample line from the pinch valve then click Next To remove grasp the sample line on both sides of the pinch valve and pull the sample line out towards you Pinch valve a ee f A Sample line Changing the Sample Line and Probe Non PEEK sample line compatible models 5 Grasp the T shaped portion of the sample line connector and gently unscrew it counterclockwise to remove it from the inlet port on the chip loader then click Next Take care when removing the sample line Sample fluid may be partially conductive or highly corrosive which can damage electronic circuits if any droplets fall down between the gaps in the sample loader cover and come into contact with internal components Push and hold the sample probe release catch to the right and pull the probe straight up and out then click Next e Take care when handling the sample probe It is a high precision component which can be damaged easily if handled carelessly e The sample line can be discarded Discard the sample line in accordance with the laboratory rules and local ordinances and regulations 7 Prepare a new sample line and sample probe attach the
327. using the checkbox beside the gate name Move to top Moves the selected gate to the top of the gate list and displays the selected color at the front on dot plots Move up Moves the selected gate up one row in the gate list and displays events with the selected color one rank towards the front on dot plots Move down Moves the selected gate up one down in the gate list and displays events with the selected color one rank towards the back on dot plots Move to bottom Moves the selected gate to the bottom of the gate list and displays the selected color at the back on dot plots Create Parent Plot Adds a parent plot to the worksheet Remove Deletes the selected gate Close Closes the dialog e Boolean Gate tab Adds a Boolean gate to the gate hierarchy For details about the Boolean Gate tab see Adding a Boolean gate to the Gate Hierarchy page 89 Tube 2 Data Source 1 Gate Editor Select Exporting statistics table dialog The Exporting statistics table dialog is displayed by clicking Export Statistics to CSV File on the Worksheet tab or Plot Tools tab of the ribbon The Exporting statistics table dialog is used to export the contents of the Gates and Statistics table as a CSV format file FITC Exporting statistics table Output Path Output Path Specifies the export destination folder Click Browse to specify for a destination folder Progress
328. velengths The highest measurement resolution is obtained when the sample fluid flow is focused into a single file stream of cells moving past the optical detection point at low sample flow pressure maximizing the exposure to excitation laser energy that each cell receives The diameter of the channel in the sorting chip narrows as it approaches the nozzle at the bottom of the chip increasing the flow speed while maintaining the relative proportion between the sheath and sample fluid Steady fluid flow ejected from the nozzle is critical for accurate sorting hence vibrations of the main unit or the sheath line on the rear panel must be avoided during sorting Fluid Flow sojdiouudg BuryessdoQ y xipueddy 193 sojdiouudg Buryeiodo y xipueddy 194 Droplet Sorting Sorting chip Breakoff point Negative deflection plate j J Nozzle Last attached droplet Satellite droplets _ Positive deflection plate O Collection tubes Waste catcher When the sorting chip is inserted in the chip loader a transducer causes the chip to vibrate at an ultrasonic frequency as fluid flows through the chip The vibrations cause the fluid flow to be ejected in a jet of uniform droplets from the nozzle at the bottom of the chip At a point a short distance beneath the sorting chip called the breakoff point the fluid flow breaks into a very regular stream of uniform droplets The size of
329. verts the selected gate to a polygon gate Copy Region Paste Region Copies the selected gate and pastes it into another plot Visible Gate Shows hides the selected gate Remove Deletes the selected plot Send to Back Moves the selected gate to the back Properties Displays the Property Window dialog page 130 Gates and Statistics table The following menu commands are displayed when right clicking within the Gates and Statistics table Show Table Shows hides the Gates and Statistics table Save as CSV File Displays the Exporting statistics table dialog page 128 for exporting the data in the Gates and Statistics table as a CS V format file Copy Picture Copies the Gates and Statistics table to the clipboard for pasting in other software for example to produce reports Open Gates Editor Displays the Gate Editor dialog page 127 to edit gates Open Statistics Editor Displays the Statistics Editor dialog page 92 to configure the statistics displayed in the Gates and Statistics table Gates and Statistics table cells The following menu commands are displayed when right clicking within a Gates and Statistics table cell Create Density Plot Adds a density plot for the events within the selected gate Create Dot Plot Adds a dot plot for the events within the selected gate Create Histogram Plot Adds a histogram plot for the events within the selected gate C
330. w sample pressure is detected while initializing the regulator Please contact your service representative Failed to move the waste tray The waste fluid tray is not operating correctly Contact your Sony distributor for service Failed to agitate the sample tube The agitation unit of the sample loader is not operating correctly Contact your Sony distributor for service Sorting error has been detected Acquisition was stopped A sorting error has occurred Check that there are no abnormal objects in the collection area Wait for a short while before restarting sorting If the problem persists contact your Sony distributor for service sno ueLjj s g xipueddy Failed to collection load Loading of the collection stage failed Check that there are no abnormal objects in the collection area Wait for a short while before loading again If the problem persists contact your Sony distributor for service 224 Error Messages Optional Accessories For details about purchasing optional accessories contact your Sony distributor DI water tank kit a U3F005 with DI water tank air filter Sheath tank LE U3F007 Sintered sheath filter LE U3F008 Waste tank LE U3F009 Waste tank air filter LE U3F011A Ethanol tank kit LE U3F012 with ethanol tank air filter Software License LGPL This product uses the following LGPL software The customer is entitled to obtain modify and redistribute the source code for th
331. xperiment data selected in the experiment data list to Selected Experiment List Clicking All gt gt adds all the experiments in Experiment List to Selected Experiment List Selected Experiment List Lists the experiments to be exported Delete selected experiments after export completed Deletes the exported experiments from the database after exporting has finished Open exported folder after export completed Opens the export destination folder after exporting has finished Remove Removes the experiment selected in Selected Experiment List from the list Clear Clears the Selected Experiment List 10 Output File Specifies the export destination file Click to specify the file Browse D Progress Displays the export progress Export Starts exporting the experiment data Close Closes the dialog Delete All Data dialog The Delete All Data dialog is displayed by clicking Delete All Data in the Database window The Delete All Data dialog is used to remove all data including user accounts and experiments e Resetting the database resets all data including user accounts experiments and analysis results e After resetting the database SH800 Software automatically shuts down Delete All Data A Are you sure you want to delete all the user and experiment data Deleted data cannot be restored The application will be automatically shutdown upon selecting
332. ydrodynamically focused sample Excitation Objective lens Detection laser module detection point module Sorting Data module acquisition Charged droplet deflection Collection Host computer module for analysis e The flow of waste fluid ethanol and DI water is not depicted in this diagram All waste fluid is collected and pumped to the waste tank in the fluidics cart Cell Sorter Block Diagram System Configuration The SH800 system comprises the SH800 main unit fluidics cart host computer and compressed air supply SH800 Software L Host computer Main unit Air compressor Fluidics cart or compressed air supply System Configuration 15 roneo was HIN MAIAMOAQ_ Je deyD 16 Name and Function of Parts Front Panel Flip up door Sorting area door button O Sorting area door Flip up door Provides access to the chip loader for inserting and removing the sorting chip The door also provides access to the optical filters and the sample line and probe for routine maintenance Make sure to close the flip up door to run samples The flip up door opens and closes with assistance from the compressed air supply Make sure the compressed air supply is turned on and supplying the rated air pressure when operating the door For safety a lock function operates to prevent the door from opening and closing when the air supply is

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