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HTS User`s Guide

1. 12 Click the Acquisition tab in the Experiment Layout Figure 3 11 and set the number of Events to Record Name Events to Record setup controls 2500000 compensation controls 10000 specimens 10000 72 BD High Throughput Sampler User s Guide NOTICE BD recommends that you set the aspirated volume for the setup controls high enough so that you do not reach the number of events before you reach the BD FACSDiva stopping time Vv Tip Press Ctrl Shift End to set multiple fields at one time and then type in the number of events in one of the fields all selected fields will reflect the new number of events Vv Tip Another way to add a value to the Events to Record field using the list is to select the field you want to change and double click the item in the list to apply it to the field See the BD FACSDiva Software Reference Manual for more ways to add events Figure 3 11 Acquisition tab of Experiment Layout Experiment Layout Labels Keywords Acquisition Quick Entry Events to Record Tih v Global Worksheet 10000 Stopping Gate All Events J Storage Gate All Events v Events to Rec x 2500000 10 000 10000000 10 000 o c8 10 000 13 Click OK to save the changes Chapter 3 Running Samples 73 74 Setting Up the Worksheet This section describes how to set up plots i
2. a ora 10 bleach solution 106 BD High Throughput Sampler User s Guide A To ensure that the 10 bleach solution retains its full germicidal effect prepare a fresh solution daily NOTICE If you wish to run BD FACSRinse solution as part of the daily cleaning procedure repeat the daily cleaning using BD FACSRinse solution in wells A1 A4 and DI water in wells B1 B4 5 Remove or open the safety cover and place the plate on the plate holder Make sure the plate corresponds to the type selected in the software Orient the plate with well A1 on the back right corner of the stage See Figure 4 4 on page 107 Verify that the sample coupler is properly installed and not leaking Figure 4 4 Orienting the plate 6 Replace the safety cover 7 BD LSR II Place the cytometer in Run mode 8 Verify that the Cytometer window displays Cytometer Connected Chapter 4 Maintenance 107 9 Click OK to dismiss the Cleaning Confirmation message Figure 4 3 on page 106 The cytometer goes through a homing sequence and cleaning begins Note that the cleaning procedure can take up to 15 minutes 10 Click OK when the completion message appears Sequence Done G HTS Clean Complete 11 Remove and discard the multiwell plate or rinse it for use on another day 108 BD High Throughput Sampler User s Guide Cytometer Inspection and Servicing Inspect the following cytometer components at the end of each day and service them
3. Turn the sample injection port wash station C slightly to disengage the magnetic force and then lift it up to expose the thumb screw D on the sample injection tubing A and the thumb screw E on the secondary pump valve tubing F Figure 4 7 Figure 4 7 Accessing the tubing sample injection port thumb screw A sample injection tubing A connector G cc thumb screw E Unscrew the thumb screw E from connector G and remove the secondary pump valve tubing F Figure 4 5 on page 116 and Figure 4 7 Chapter 4 Maintenance 117 e Disconnect the sample injection tubing A from the sample injection port wash station C by unscrewing the thumb screw D Figure 4 7 and remove the sample injection tubing A 6 Discard the sample injection tubing A and the secondary pump valve tubing F as biohazardous waste 7 Install new sample injection tubing Replacement tubing is included in the accessory kit e Screw the thumb screw D onto the sample injection port wash station C e Screw the thumb screw E from the secondary pump valve tubing into connector G NOTICE Take care to screw the thumb screws on straight If you attach them at an angle you could strip the threads 8 Insert the secondary pump valve tubing F into the opening of the cytometer base and route the tubing to the secondary pump Then lower the sample injection port wash station C onto the cytometer base plate The magnet
4. rr oO Tia lia See ee By 2 Acquisition Status Toia A Wash Volume L 200 BB Processed Events Electronic Abort Rate Threshold Count Electronic Abort Count Demo BD High Throughput Sampler User s Guide The following table describes some of the more common workspace components specific to running the HTS Use the commands in the menu bar to operate the software File Edit View Experiment Populations Worksheet Instrument Carousel HTS Help e Use the Experiment menu to add a new experiment to the Browser or open a template A new experiment automatically contains global cytometer settings and a global worksheet For information see Creating Experiments on page 64 e Use the commands in the HTS menu to perform maintenance procedures on the HTS For information see Maintenance on page 103 H Click a button in the Workspace toolbar to hide or show the corresponding window Windows can be resized by dragging a border or corner Click the plate button to view the Plate window e Use the Browser window to create and set up experiments and view experimental data hierarchically In the Browser double click an experiment to open it e Click the New Experiment button fj in the Browser toolbar to create a new experiment e Click the New Plate button in the Browser toolbar to add a default 96
5. 2 Choose HTS gt Prime 3 Verify that the pumps are primed and the HTS tubing fills with sheath fluid Once priming is complete the following message appears G HTS Prime Complete 4 Click OK to dismiss the message Performing a Motion Test You might need to perform a motion test when you set up the HTS unit for the first time ie after a depot repair procedure when axis movement is suspect or when you are directed to do so by a BD Biosciences service representative during cytometer troubleshooting 138 BD High Throughput Sampler User s Guide Note that this test will execute the same sequence whether a 96 or 384 well plate is selected For the most accurate troubleshooting use a 384 well plate to verify the mechanism reaches the extreme positions 1 Choose HTS gt Motion and Position Test The Motion and Position Test dialog appears 2 Choose the plate type from the drop down menu Motion And Position Test Motion Test 96 Vell Flat Bottom 96 Vell U Bottom 96 Well Y Bottom 384 Well Flat Bottorn After you select the plate type the Motion Test button _ ation test_ becomes enabled 3 Install the corresponding plate onto the plate holder replace the safety cover and click Motion Test Mationtest 4 Verify that the sample probe properly performs the following homing sequence e The probe travels to the home position e The probe moves up and then moves to the ex
6. The following dialog appears Please replace sheath solution tf Cancel 7 Click OK 8 Click OK in the next dialog to confirm the duration A dialog appears informing you that the sheath exchange is complete Sheath Exchange Status wD Fluidics sheath exchange is complete 9 Click OK NOTICE When the sheath type is BD FACSFlow the sheath fluidics level indicator will be green When the sheath type is FACS sheath solution with surfactant the level indicator will be white Chapter 3 Running Samples 99 NOTICE If you exchanged BD FACSFlow with BD FACS sheath solution with surfactant install the HTS sample coupler then prime the HTS twice by choosing HTS gt Prime This will exchange the fluid in the syringes with BD FACS sheath solution with surfactant Shutting Down Perform a daily cleaning procedure before shutting down the system 1 2 5 6 Run a daily cleaning procedure see Daily Cleaning on page 104 BD FACSCanto and BD FACSCanto II Perform a fluidics shutdown Refer to Fluidics Shutdown in the following section BD LSR II Prime the HTS unit Place the cytometer in Run mode Choose HTS gt Prime When priming is complete click OK in the dialog then place the cytometer in Standby mode NOTICE To prime the cytometer press the Prime button on the fluid control panel on the cytometer itself BD FACSCanto and BD LSR II Turn off the power to the HTS Turn off the
7. NOTICE If you launched BD FACSDiva software when the HTS was switched off it might take a few minutes to complete the initialization of the loader 6 Select the two setup control well in the plate layout of the Setup view 1 The well is outlined in green El Chapter 3 Running Samples 79 7 Click _Grunwes in the Acquisition Dashboard If necessary choose View gt Acquisition Dashboard The HTS homes and primes the probe and then aspirates the unstained control from the first setup control well NOTICE Setup and compensation wells are always acquired in standard mode even if high throughput mode is selected in the Plate window 8 Adjust FSC SSC threshold and PMTs using the unstained control For more information on optimizing cytometer settings refer to the appropriate cytometer user s guide NOTICE Acquisition stopping time is determined by BD FACSDiva software according to sample volume sample rate BD recommends you set the target number of events high enough so that you do not run out of events before you reach the software based acquisition stopping time For more information on stopping time see Running Samples Automatically in Sequence on page 46 and Running Wells Manually on page 47 Mi Tip If you run out of sample before you finish optimizing settings allow the HTS to move to the next setup control well and continue optimization from there 9 Click Stop Well s _ sweis once you have optimized setting
8. 90 Figure 3 18 Keyword Analysis view showing Granulocytes wells Plate 96 Well U bottom Setup Analysis Legend Keywords meg Specimen type bE First well in group b Specimen number Name Suffix BB Concentration E Lymphocytes D rne a List of specimens on the plate Ea Setup Controls _001 E Compensation Controls E3 Specimen_001 4 Specimen_002 11 Print the Granulocytes Keyword view and legend Performing Data Analysis Analyze plate data in the same manner you would analyze tube data using BD FACSDiva software For detailed instructions refer to the BD FACSDiva Software Reference Manual Performing Batch Analysis Do the following to set up batch analysis 1 Open an experiment containing the plate data that you want to batch analyze 2 Double click the plate in the Browser to open the Plate window BD High Throughput Sampler User s Guide 3 Click the Analysis tab to display the Analysis view 4 Inthe global worksheet verify that gates are adjusted to enclose the appropriate populations 5 Inthe plate layout select a well or wells for batch analysis right click to open the shortcut menu and then choose Batch Analysis NOTICE If you wish to print you must assign a printer before starting batch analysis E Plate 96 Well U bottom Setup Analysis Legend Keywords wy Specimentype E
9. Figure 4 13 Installing the syringe valve gt syringe plunger drive plunger lock screw __ gt 12 If you e replaced the primary pump syringe skip to step 13 e replaced the secondary pump syringe reconnect the HTS to the cytometer by following the procedure in Installing the HTS Unit on page 170 13 Refill the pump e BD LSR II Verify that the cytometer is still in Run mode Turn on the BD FACSCanto II e Reattach the sheath line and filter in back of the HTS unit e BD LSR I and BD FACSCanto Turn on the HTS e BD LSR II Choose HTS gt Prime to prime the fluidics until the plunger barrel is full of sheath fluid If you see any bubbles continue priming until the bubbles are gone BD FACSCanto and BD FACSCanto II Choose Cytometer gt Fluidics Startup to perform a fluidics startup Verify that the plunger barrel is full of sheath fluid If you seen any bubbles choose HTS gt Prime repeat until all bubbles are gone 128 BD High Throughput Sampler User s Guide Replacing the Probe The probe is located at the end of the probe assembly arm Replace the probe whenever it is bent or permanently clogged A spare probe is included in the accessory kit A All cytometer surfaces and hardware that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when replacing the probe Wear suitable protective clothing eyewear and gloves Fig
10. Name Suffix List of specimens on the plate H Setup Controls_001 EZ Specimen_001 3 Specimen_002 Compensation Controls 2 Click the Add Keywords button A in the Plate toolbar The Add Keywords dialog appears See Figure 3 15 on page 87 86 BD High Throughput Sampler User s Guide Figure 3 15 Add Keywords dialog Available Keywords Selected Keywords a PATIENT ID CASE NUMBER SOP SINST ney es GUID PLATE NAME PLATE ID WELL ID Concentration Lymphocytes Granulocytes zi 3 Alt Shift Click the keywords Concentration Lymphocytes and Granulocytes from the Available Keywords list on the left side of the dialog The Concentration Lymphocytes and Granulocytes keywords were created for this example 4 Click Add gt to move the keywords to the Selected Keywords column click OK The three keywords now appear in the Keywords list in the Analysis view 5 Click the keyword Concentration in the Keyword list The Analysis view changes to the Keyword view Figure 3 16 on page 88 If values were assigned to the selected keyword the values are displayed in this view In this example the selected keyword is Concentration and the values are 0 10 0 25 0 50 and 0 75 Chapter 3 Running Samples 87 Figure 3 16 Keyword Analysis view showing Concentration values Plate 96 Well U bottom Setup Analysi
11. Sample volume too large Decrease the sample volume Wash volume too low Increase the wash volume Sample coupler not completely installed Slide sample coupler on SIT until you reach a hard stop Damaged injection tubing Replace the sample injection tubing See Replacing the Sample Injection Tubing on page 116 148 BD High Throughput Sampler User s Guide BD FACSDiva Troubleshooting Observation System error Possible Causes Cover error Recommended Solutions Replace cover and resume run Back pressure error e Ensure waste line to cytometer is properly installed and not kinked e Ensure filter on waste tank is not clogged Motion error Select HTS gt Home and resume run Pump error Select HTS gt Home and resume run Plots not refreshing Incorrect worksheet displayed Ensure global worksheet is displayed during a sequenced plate based acquisition Run buttons not enabled No global worksheet selected Ensure a global worksheet is selected in the global worksheet folder Well s not selected Select a well s BD FACSCanto and BD FACSCanto II only HTS not included in cleaning modes Sample coupler not installed Ensure sample coupler is installed before cleaning modes are selected No power Ensure HTS is powered on Cover open Close cover Chapter 5 Troubleshooting 149 150 BD High Throughput Sampler User s
12. e Pull the sample coupler down and away from the SIT leaving the nut attached to the sample coupler Locate and loosen the positioning screw that secures the HTS unit to the base plate See Figure B 1 on page 160 Appendix B Depot Repair Procedures 159 Figure B 1 Location of positioning screw example of BD LSR II base plate positioning screw 6 Grasp the sides or back of the HTS unit and slide it forward until it meets the stop in the base plate 7 BD FACSCanto II Remove the probe refer to Replacing the Probe on page 129 and the overflow reservoir A Any cytometer surface that comes in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling cytometer hardware Wear suitable protective clothing and gloves 8 BD FACSCanto Remove the catch tray 9 BD LSR II and BD FACSCanto Lift the HTS unit off of the base plate by doing the following A To prevent personal injury or damage to the HTS unit do not slide the HTS unit out so far that it becomes unbalanced e Slightly lift the HTS unit to clear the positioning screw e Being careful not to strain the sheath tubing slide the unit towards you until you can access the filter 160 BD High Throughput Sampler User s Guide 10 11 12 13 14 This allows you better access to the connections on the back of the unit Disconnect the sheath and waste lines from the cytometer BD LSR II and BD FAC
13. All other company and product names might be trademarks of the respective companies with which they are associated Patents PerCP US 4 876 190 APC Cy7 US 5 714 386 FCC Information WARNING Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment NOTICE This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his or her own expense Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits This Class A digital apparatus meets all requirements of the Canadian Interference Causing Equipment Regulations Cet appareil num rique de la classe A respecte toutes les exigences du R glement sur the mat riel brouilleur du Canada Notice BD Biosciences delivers software and workstations that are intended for running the cytometers s
14. Chapter 4 Maintenance 115 Replacing the Sample Injection Tubing The sample injection tubing connects the injection port wash station to the secondary pump valve tubing Replace the tubing every 6 months Figure 4 5 Disconnecting sample injection tubing from injection port wash station sample injection tubing thumb screw hex screw A secondary pump valve tubing sample injection port connector wash station oO thumb screw A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when changing tubing Wear suitable protective clothing and gloves 1 Perform the monthly cleaning procedure to decontaminate the sample injection port wash station See Monthly Cleaning on page 111 2 Turn off the HTS power switch BD LSR II and BD FACSCanto or turn off the cytometer BD FACSCanto II 3 For easy access remove the HTS unit See Removing the HTS Unit on page 159 for instructions 4 Move the probe arm up and then push it toward the front of the HTS 116 BD High Throughput Sampler User s Guide 5 Remove the sample injection tubing A from the HTS If necessary disconnect the top hex screw B from the secondary pump using the 5 16 in wrench in the accessory kit Figure 4 5 on page 116 and Figure 4 6 Figure 4 6 Hex screw on secondary pump hex screw B secondary pump secondary pump valve tubing F
15. Generic APC Generic e APC Cy Generic 8 Click OK The specified compensation controls are added to wells B1 B7 9 Click and drag to select the following wells and then click the Add Specimen Wells button AJ to add two specimens to the plate layout Wells Name C1 C12 Specimen_001 D1 D12 Specimen_002 Mi Tip You can change the specimen name using the Specimen Inspector Inspector Specimen_001 Specimen Keywords Cytometer Settings Name Specimen _001 Collected Global Worksheet wa 70 BD High Throughput Sampler User s Guide The plate layout should look similar to Figure 3 9 Figure 3 9 Setup view E Plate 96 Well U bottom Setup Analysis Filter Setup Details Plate Information a F i i Specimen type Pej Acquisition order Specimen settings Throughput Mode High Standard bE First welin group b Specimen number Well settings Plate Status Loader Status List of specimens on the plate Setup Controls _001 EE Compensation Controls Specimen_001 Loader Settings Sample Flow Rate uL sec 10 Sample Volume uL 10 i Mixing Volume uL 100 EF Mixing Speed yL sec 180 SF Number of Mixes 2 Wash Volume uL 400 SF 10 Choose Experiment gt Experiment Layout and click the Labels tab Figure 3
16. Table 1 3 Volume Type Volume Type Definition Well volume Total volume Aspirated excess volume Available volume Minimum volume Mixing volume Dead volume Volume that well can hold filled to the brim Volume pipetted into well aspirated excess volume Standard mode 20 uL Volume pipetted into well aspirated excess volume dead volume 50 uL for both standard and high throughput modes for 96 well plates Volume one half the available volume NOTICE A mixing volume that is larger than the available volume introduces air bubbles into the sample Volume in the bottom of the tube that the probe cannot reach Chapter 1 Introduction 25 26 Mixing HTS mixing efficiency is impacted by the viscosity of the sample in the well Adjust the mixing volume speed and the number of mixes to obtain the most homogeneous population for acquisition A greater number of mixes might allow you to decrease the mixing volume and speed but this could impact sample throughput Increasing the mixing speed and volume might improve throughput and mixing efficiency but it could also introduce air bubbles in the sample well In addition increased mixing speed could compromise the separator bubble between the sample and sheath resulting in sporadic event rates and possibly higher carryover Use default values as a starting point see Table 2 4 on page 40 with a mixing volume of one half the total sample volume No
17. You cannot change HTS settings during acquisition or when a sequence is in process NOTICE If multiple wells with different loader settings are selected a red highlight appears around the loader settings that are different Loader Settings Sample Flow Rate uL sec Sample Volume uL Mixing volume uL Mixing Speed uL sec Number of Mixes Wash Volume uL BD High Throughput Sampler User s Guide 10 3 le la 50 fz 200 SF v 0 KB Table 2 3 provides a description of each setting Table 2 3 HTS settings definitions The Sample Flow Rate is the speed the syringe aspirates sample from the well in pL second To determine the acquisition time divide the sample volume by the sample rate sample volume uL sample rate uL sec acquisition time sec The Sample Volume is the volume of sample aspirated from each well for acquisition During acquisition in standard mode BD HTS aspirates the selected sample A volume 2 200 pL plus an additional 20 pL from the well During acquisition in high throughput mode BD HTS aspirates a fixed 22 pL per well even though you can select a sample volume between 2 10 pL Make sure each well contains sufficient sample for the entered volume plus the A dead volume Insufficient volume can introduce air bubbles into the system Mrip To make sure you do not run out of sample BD recommends that you prepare your plate with a minimum of 250
18. as needed A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling cytometer components Wear suitable protective clothing eyewear and gloves e Sample coupler Check for leaks around the sample coupler on the cytometer SIT If necessary tighten the top nut to secure the sample coupler to the SIT BD LSR Il BD FACSCanto BD FACSCanto II tightening nut If the coupler continues to leak after you tighten the fitting remove and then reinstall the coupler as follows Hold the coupler with one hand while you unscrew the tightening nut with the other hand When the coupler is loose pull it straight down Chapter 4 Maintenance 109 Slide the coupler back up until you reach a hard stop Make sure the sample coupler tubing is not kinked or twisted Hold the coupler with one hand while you tighten the tightening nut with the other hand If the coupler is still leaking replace it See Replacing the Sample Coupler and Tubing on page 123 e Injection port wash station Check this area for residual liquid Liquid appearing around the injection port wash station may indicate a clog If you suspect a clog refer to HTS Troubleshooting on page 146 e Base plate probe arm assembly and plate holder Dampen a clean lint free cloth with distilled or DI water and wipe down only the black surfaces of these components as needed
19. e Includes two throughput modes standard and high throughput e Minimizes carryover e Provides user definable mixing and sample introduction protocols e Is user installable software and sampler unit only initial installation excluded e Supports immunophenotyping assays The HTS can process a 96 well plate in approximately 44 minutes in standard mode and approximately 15 minutes in high throughput mode using the default settings listed in Table 1 1 Table 1 1 Default throughput mode settings Setting S eee Sample Flow Rate pL sec 1 1 Sample Volume pL 10 2 Mixing Volume pL 100 50 Mixing Speed pL sec 180 200 Number of Mixes cycles 2 2 Wash Volume pL 400 200 Approximate Acquisition Time min 44 15 14 BD High Throughput Sampler User s Guide For more information see Loader Settings on page 38 The HTS unit is installed on the cytometer by a BD Biosciences service engineer Once installed the HTS enables quick conversion of the flow cytometer from tube to plate based acquisition Figure 1 1 HTS installed on a BD LSR II flow cytometer BD LSR II flow cytometer BD HTS Unit Chapter 1 Introduction 15 Components See Figure 1 2 on this page Figure 1 3 on page 17 and Figure 1 4 on page 18 to familiarize yourself with the specific HTS hardware components for your cytometer For a description of HTS cytometer connections see Cytometer Connections on page 19 F
20. e Sample probe Make sure the probe is straight If it is bent or shows signs of wear replace the probe See Replacing the Probe on page 129 e Pumps Wipe up any spills on or around the pumps Inspect the fittings to make sure they are tight See Inspecting Thumbscrew Fittings on page 142 and Inspecting Hexagonal Fittings on page 143 Inspect the pump syringes for leakage If your system has been used extensively and the syringes appear worn replace them See Replacing a Pump Syringe on page 125 e BD LSR II and BD FACSCanto Absorbent pad Inspect the absorbent pad at the back of the HTS unit to make sure it is not saturated Replace the pad if needed 110 BD High Throughput Sampler User s Guide Monthly Maintenance When running the HTS daily perform the following cleaning procedures at the end of every month If you are using the HTS less frequently adjust the monthly maintenance schedule accordingly e Monthly Cleaning see the following section e Surface Inspection and Cleaning see page 114 Monthly Cleaning BD LSR II Monthly Cleaning To perform a monthly cleaning procedure choose HTS gt Monthly Clean The software prompts you to install a tank containing BD FACSClean or a 10 bleach solution in place of the cytometer sheath tank and then pumps cleaning solution through the lines for 30 minutes When cleaning is complete the software prompts you to install a rinse tank and then pumps DI water thro
21. well U bottom plate to the open experiment e Click the arrow next to the New Plate button gj EI to choose a new plate type to add to the experiment The type you choose becomes the default Browser foL Experiment_001 toolbar f g se p 2 al r FE B 96 well U bottom plate F a i Name Date 96 well V bottom plate B Administrator Foker 001 96 well flat bottom plate open Hd Experiment _001 1 18 07 3 31 36 PM 384 well flat bottom plate experiment i E Cytometer Settings p Global worksheets current tube 53 96 Well U bottom f s p pointer ag 96 Well bottom S i Blank Experiment with Sam 1 23 07 10 04 18 AM closed 1B shared view experiment Chapter 2 BD FACSDiva Software Overview 29 Use the Acquisition Dashboard to acquire and record well data Use the HTS controls to acquire and record wells in sequence using the selected throughput alg mode Run Plate runs the wells from the current position to the end of the plate Run Well s runs the selected wells only Use the Basic Controls to manually acquire or record selected wells in standard mode using the current loader settings EH Acquisition Dashboard Current Activity Active TubeWell Threshold Rate Stopping Gate Events Elapsed Time AT 0 evtis Oevt 00 00 00 Basic Controls oJ Next Tut OB Acquire Data OB Record Data Plate Controls Run Plate TE Run wells iut Acquisition S
22. 10 on page 72 11 Define fluorophore labels for both samples Fluorophore Label Fluorophore Label FITC CD14 PE Cy7 CD19 PE CD16 CD56 APC CD3 PerCP CyS 5 CD8 APC Cy7 CD4 e Select the FITC column of Specimen_001 and Specimen_002 e Click in the Label field and enter CD14 The selected fields are labeled CD14 e Label the remaining fluorophores Chapter 3 Running Samples 71 Vv Tip There are other fast ways to create labels Refer to the BD FACSDiva Software Reference Manual The Experiment Layout should look similar to Figure 3 10 Figure 3 10 Labeled fluorophores in Experiment Layout Experiment Layout Labels Keywords Acquisition Quick Entry Labels Label Name List by user Administrator Specimen_001 H BD Defined Name Label PE PerCP Cy5 5 APC Cy7 ec CD16 CD56 CD8 CD4 PE PerCP Cy5 5 APC Cy7 CD16 CD56 CD8 CD4 PE PerCP Cy5 5 APC Cy7 CD16 CD56 CD8 CD4 PE PerCP Cy5 5 APC Cy7 CD16 C056 CD8 CD4 PE PerCP Cy5 5 APC Cy7 CD16 CD56 CD8 CD4 PE PerCP Cy5 5 APC Cy7 CD16 CD56 CD8 CD4 PE PerCP Cy5 5 APC CY7 CD16 C056 CD8 CD4 PE PerCP Cy5 5 APC CY7 CD16 C056 CD8 CD4 PE PerCP Cy5 5 APC Cy7 CD16 CD56 cbs cD4 PE PerCP Cy5 5 APC Cy7 Addt CD16 CD56 CD8 CD4 o List Delete from List PE PerCP Cy5 5 APC Cy Assign or Remove Labels CD16 CD56 CD8 CD4 PE PerCP Cy5 5 APC Cy h amp
23. 119 shown 17 18 fluid exchanging 96 98 for use on HTS 56 shipping packaging 175 soft standby mode 23 software application window shown 28 batch analysis 13 90 main features 27 menus 29 quitting 101 troubleshooting 149 specimen renaming 42 specimen copying and pasting 42 speed mixing 39 standard mode 23 37 status indicators well colors 35 stopping HTS 84 software 101 syringes replacing 125 T technical assistance xi templates cleaning 105 experiment 84 panel 85 plate 76 test motion 138 throughput mode 37 optimizing 26 thumbscrew fittings inspecting 142 time acquisition 39 total volume 25 troubleshooting acquisition 147 hardware 146 homing the sample probe 136 leaking valves 124 133 143 160 software 149 suggestions 145 149 tube mode 19 94 tube based acquisition returning to 94 safety 95 vs plate based 22 tubing replacing 116 123 thumbscrew fittings 142 U unscheduled maintenance 136 144 user preferences 65 182 BD High Throughput Sampler User s Guide V views Acquisition Dashboard 30 Analysis 48 Browser 29 Inspector 31 plate layout 35 Plate window 31 Setup 33 volume minimum sample 39 mixing 39 sample 39 volumes choosing loader settings 25 types 25 well 25 W wash station shown 17 18 wash volume probe 39 wells apply analysis template 40 apply application settings 40 apply cytometer settings 40 border colors and status 35 compensation setup 40 copying
24. Calculate Compensation If the calculation is successful a dialog appears Chapter 3 Running Samples 81 Mi Tip Name the compensation setup after the experiment and click OK For information on performing compensation refer to the BD FACSDiva Software Reference Manual Compensation controls can be run individually through their normal worksheets using Basic Controls in the Acquisition Dashboard Acquiring Data 82 Once you calculate compensation you are ready to record your samples 1 2 Click the Worksheets View button to return to the global worksheet Select well C1 in the plate layout Click Run Plate _ runiete in the Acquisition Dashboard Wells will be acquired in the order they were created As each well is injected into the cytometer SIT events appear in plots on the global worksheet If an error occurs an error message will appear and the well in the plate layout will be colored according to its status Well status is indicated by the color outlining each well as described in Plate Layout on page 35 The HTS is equipped with a safety interlock that prevents the cytometer from running when the safety cover is removed or opened Do not remove or open the safety cover while samples are being processed To pause or stop acquisition click the corresponding buttons in the Acquisition Dashboard before you remove or open the safety cover At the end of the run a dialog appears indicating the run is com
25. Figure 2 2 Setup view of Plate window view tabs plate toolbar Plate 96 Well bottom Setup Analysis Filter Setup Details m a Specimen type r Acquisition order 7 Specimen settings hE First wellin group 2 Specimen number a 7 Well settings PQs isl 3 4 plate legend filter Plate Information Throughput Made High Standard i throughput mode loader status Plate Status Idle List of specimens on the plate specimen list Loader Settings Sample Flow Rate iL sec 108 Sample Volume uL E Mixing Volume uL BEIN Mixing Speed uL sec 200 SF HTS settings Number of Mixes 20 Wash Volume uL 200 SA plate layout See the following sections for a descriptio functionality of each view n of components in and the 32 BD High Throughput Sampler User s Guide Setup View Components The Setup view contains controls for designing experiments running plate based acquisition and monitoring acquisition status For instructions on setting up an experiment see Creating Experiments on page 64 All entries made in the Setup view can be saved as a template either for general projects or as a default experiment for a user defined project The following components are available in the Setup view Plate Filter The Filter Setup Details area provides a legend indic
26. Guide Appendix A Consumables and Replacement Parts This appendix provides a list of supplies and replacement parts for your HTS unit To order spare parts and consumables from within the US call 877 232 8995 or go to bdbiosciences com In other countries contact your local BD Biosciences representative This information is correct at the time of publication For up to date information refer to our website bdbiosciences com You will find the following in this appendix e Cytometer Supplies on page 152 e Maintenance Log on page 154 151 Cytometer Supplies The HTS is shipped with a preventive maintenance kit including replacement parts for one year of maintenance and an accessory kit containing other replacement parts Use the following part numbers if you need to order part replacements Note that parts in the preventive maintenance kit can also be ordered individually Table A 1 Preventive maintenance kit part no 337042 Item Part No Kit Quantity Glass syringe 500 pL 344912 2 Tubing assembly secondary pump to cytometer 335452 1 BD LSR II Tubing assembly secondary pump to cytometer 339340 1 BD FACSCanto and BD FACSCanto II Tubing assembly probe to primary pump 335454 1 Tubing assembly injection port 335526 2 Probe and tubing assembly 34389017 1 HTS sheath filter 2 335710 2 Quick disconnect O ring 343618 7 O ring sleeve retainer BD LSR II 343620 1 Air filt
27. Run setup controls to optimize cytometer settings for sample type Waste tank back pressure e Ensure waste fittings are connected and not damaged e BD LSR II Ensure waste lines are not kinked e Ensure antifoam concentrate is added to waste tank Chapter 5 Troubleshooting 147 Acquisition Troubleshooting continued Observation Unexpectedly low event rate or no events in plots continued Possible Causes Cytometer malfunction Recommended Solutions e Manually run a tube of beads and verify events appear in plots e Check the sample voltage BD FACSCalibur e Check the Bal seal Bubble in sample well s Gently tap plate to ensure all wells are bubble free then reacquire sample Bubbles in flow cell Ensure BD FACS sheath solution with surfactant is used during plate based acquisition Insufficient sample or no sample in well Ensure there is sufficient sample in well Bubbles in syringe Prime unit Choose HTS gt Prime No filter cap BD FACSCanto and BD FACSCanto II Replace filter cap Unexpectedly low number of events Insufficient stopping criteria Make sure to set the target number of events high enough that you do not recorded run out of events before you reach the software based acquisition time sample volume sample rate High carryover Mixing volume too great Decrease mixing volume Too many mixes Reduce number of mixes
28. Text and keyboard conventions are shown in Table 2 Table 1 Hazard symbols Symbol Meaning Caution hazard or unsafe practice that could result in material damage data A loss minor or severe injury or death A Risk of electric shock Biological risk a Although these symbols appear in color on the cytometer they are in black and white throughout this user s guide their meaning remains unchanged Table 2 Text and keyboard conventions Convention Use NOTICE Describes important features or instructions M Tip Highlights features or hints that can save time and prevent difficulties Italics Italics are used to highlight book titles and new or unfamiliar terms on their first appearance in the text gt The arrow indicates a menu choice For example choose File gt Print means to choose Print from the File menu Ctrl X When used with key names a dash means to press two keys simultaneously For example Ctrl P means to hold down the Control key while pressing the letter p x BD High Throughput Sampler User s Guide Technical Assistance For technical questions or assistance in solving a problem e Read the section of the user s guide specific to the operation you are performing e See Chapter 5 Troubleshooting If additional assistance is required contact your local BD Biosciences technical support representative or supplier When contacting BD Biosciences have the following
29. and default cytometer settings e Use the Experiment gt New Experiment command to create a new experiment based on a saved template with customized elements such as plots gates statistics view and cytometer settings The following tutorials describe how to set up an experiment specifically for plate based acquisition and how to use each of the above commands to create additional experiments Creating a Folder and an Experiment This section describes how to create a folder and an experiment containing basic analysis options 1 Click the corresponding buttons in the Workspace toolbar to display the Browser 6 Cytometer Inspector P Worksheet and Acquisition Dashboard l windows as needed Mi Tip As you work in the software windows can become hidden Bring a window to the forefront by double clicking the corresponding button in the Workspace toolbar 2 Choose Edit gt User Preferences and verify that the options in Figure 3 6 on page 65 in the General tab are selected 64 BD High Throughput Sampler User s Guide Figure 3 6 User Preferences dialog User Preferences Gates Worksheet Plot FCS Templates Statistics Biexponential C Tube specific worksheet C Start acquisition on pointer change Show file identifier GUID in statistics view Remove tube specific cytometer settings on duplicate M Save analysis after recording through global worksheet
30. at the front of the HTS skip to step 7 e the secondary pump syringe located at the back of the HTS remove the HTS by following steps 1 through 13 in Removing the HTS Unit on page 159 Chapter 4 Maintenance 125 Figure 4 11 Primary and secondary pumps primary syringe pump secondary syringe pump 6 Push the syringe plunger up to the top of the syringe barrel 7 Loosen the plunger lock screw approximately three full turns counterclockwise Figure 4 12 on page 127 8 Lower the plunger drive by pushing down on the plunger lock screw See Figure 4 12 on page 127 126 BD High Throughput Sampler User s Guide Figure 4 12 Lowering the plunger valve plunger drive plunger lock screw 9 Unscrew the syringe from the valve Grasp the metal ring at the top of the syringe and turn counterclockwise 10 Discard the syringe into a biohazardous sharps container 11 Install the new syringe Figure 4 13 on page 128 e Screw the syringe into the valve see a in Figure 4 13 Screw the syringe into the valve port until it contacts the seal washer and then turn the syringe an additional 1 6 to 1 4 turn If leakage is observed tighten a maximum of an additional 1 8 turn e Pull down the syringe plunger until it meets the plunger drive see b in Figure 4 13 e Tighten the plunger lock screw see c in Figure 4 13 on page 128 NOTICE Make sure the plunger lock screw is securely tightened Chapter 4 Maintenance 127
31. by clicking the corresponding mode button in the Setup view You can also select throughput mode in the Plate Inspector Throughput Mode High C Standard Default acquisition settings and limits change when you switch between standard and high throughput mode so choose the mode first when setting up a plate NOTICE The throughput mode applies to the entire plate so all wells on the plate are acquired in the same mode except for setup and compensation control wells which are always run in standard mode The following acquisition times can be achieved with the settings specified in Table 1 1 on page 14 e In high throughput mode the HTS can process a 96 well plate in approximately 15 minutes e In standard mode the HTS can process a 96 well plate in approximately 44 minutes For details on how the HTS acquires samples in each mode see Sample Processing on page 23 Loader Status Monitor status messages for the HTS Loader during acquisition by viewing the Loader status field For Loader errors see HTS Troubleshooting on page 146 Chapter 2 BD FACSDiva Software Overview 37 38 Loader Settings The loader HTS settings shown in the Setup view are the current settings for the selected well or plate You can modify these settings in either the Setup view or the Well Inspector Default loader settings are provided for each throughput mode You will need to optimize these settings for the plate type and assay you are running
32. cell and a new cycle begins after the last well in a sequence raises the probe to the cleaning position and performs a final flush and refill lowers the probe into the injection port wash station moves the probe up over and down into the injection port e injects the sample and boosts it to the cytometer flow cell at high speed e anon selectable fixed boost command transports sample to the flow cell to prime the sample flow path e delivers the sample to the flow cell at the required rate and volume using the secondary high throughput pump e simultaneously raises the probe to the cleaning position and the primary pump begins processing the next well in parallel to acquisition once the secondary pump delivers the sample pushes any remaining sample and the separator bubble through the flow cell The next well s sample is injected and a new cycle begins after the last well in a sequence raises the probe to the cleaning position and performs a final flush and refill lowers the probe into the injection port wash station BD High Throughput Sampler User s Guide Understanding Volumes There are different volumes to take into consideration when choosing loader settings Figure 1 9 shows different well volumes and Table 1 3 defines each volume type Figure 1 9 Well volumes sample probe total volume 4 L available volume plate dependent dead volume well
33. experiment To assign new keyword see Assigning Keywords on page 77 To view keywords in the Analysis view you must add the Keywords to the Selected Keywords list For complete instructions see Displaying Keywords on page 86 Figure 2 4 Add Keywords dialog box Available Keywords Selected Keywords Se PATIENT ID CASE NUMBER OP SINST POY ae GUID PLATE NAME PLATE ID WELL ID Concentration Lymphocytes Granulocytes Le OK Print Button Click the Print button a to print the Analysis view To save a record of analysis print this view after analysis is complete The printout shows the plate layout legend with keywords and plate name and type Keywords List The Keywords list contains the selected keywords for all samples and wells on the plate Figure 2 3 A maximum of 15 keywords can be added to the list Add keywords to the list using the Add Keyword button See Add Keyword Button for more information 50 BD High Throughput Sampler User s Guide Current Tube Pointer To analyze a well in the Analysis view click the well to select it The well is outlined in blue indicating the well is selected for analysis In the Browser the current tube pointer appears to the left of the plate name See Figure 2 5 for an example Figure 2 5 Analysis indicators BP late 96 Well U bottom a I i batiam F2 Browser Experiment _001 Setup Analysis m Legend Ga lt Fit ay Ba Specimen t
34. it completely before you reinstall it 5 Place the clean filter inside the filter retainer 6 Reinstall the filter retainer Center it over the fan guard and push evenly on the opposite edges to snap it into place 7 Place the HTS unit right side up 8 Reinstall the unit on the cytometer For detailed instructions see the Installing the HTS Unit on page 170 Replacing the Sample Coupler and Tubing If you frequently remove the sample coupler to run the cytometer in tube acquisition mode you will need to replace the coupler yearly or if the coupler continues to leak after you have tightened the fitting A All cytometer surfaces and hardware that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when changing tubing Wear suitable protective clothing eyewear and gloves Chapter 4 Maintenance 123 1 Remove the HTS unit from the cytometer For detailed instructions see Removing the HTS Unit on page 159 2 Detach the sample coupler tubing Unscrew the tubing fitting from the secondary pump valve Figure 4 10 and discard the sample coupler and tubing as biohazardous waste Figure 4 10 Detaching the sample coupler and tubing 3 Install a new sample coupler and tubing Replacement tubing is included in the accessory kit Connect the new tubing fitting to the port in the secondary pump valve Tighten the tubing fitting until it is finger tight A Valves contain se
35. of the flow direction label each quick connector so you know which end attaches to the cytometer C and which end attaches to the HTS unit 5 Remove the sheath line Press the metal tabs to release the two off white quick connectors one on the cytometer interface panel and the other on the back of the HTS unit 6 Remove the filter Detach the filter from one tubing segment by holding the filter while you twist the luer connection Repeat this operation for the other tubing segment 120 BD High Throughput Sampler User s Guide 7 Install the new filter e Attach the tubing segment with the male connector to the female filter connection Hold the filter while you twist the luer lock ring e Attach the tubing segment with the female connector to the male filter connection Hold the filter while you twist the luer female connector 8 Reinstall the sheath line between the cytometer interface panel and the HTS Attach the quick connectors at each end NOTICE Although the sheath filter is a screen filter that provides identical performance regardless of the flow direction BD recommends that you always install the sheath line with the lock ring end toward the cytometer panel connector labeled C This will prevent any foreign material accumulated on one side of the filter from flushing into the HTS if the filter is reversed 9 Reinstall the HTS unit BD FACSCanto Install the rear catch tray by routing the sample coupler un
36. pL of sample well for a 96 well plate in standard mode 100 pL well for a 96 well plate in high throughput mode and 50 pL well for a 384 well plate either mode The Mixing Volume is the volume of sample aspirated and dispensed during mixing Make sure each well on your plate contains sufficient sample for mixing A Insufficient volume can introduce air bubbles into the system BD recommends a mixing volume that is one half the available volume See Understanding Volumes on page 25 and Mixing on page 26 Adequately mix the sample before pipetting it into the plate Use the mixing A cycle only to maintain a homogeneous particle suspension The Mixing Speed is the speed that the syringe aspirates sample from and dispenses sample to the well during mixing The Number of Mixes is the number of mixing cycles that are performed before a sample is aspirated The Wash Volume is the volume of sheath fluid dispensed for rinsing between wells Chapter 2 BD FACSDiva Software Overview 39 Table 2 4 shows default HTS Settings default and range for each throughput mode Table 2 4 HTS settings for standard and high throughput mode Setting Standard Mode High Airgas Default Range Default Range Sample Flow Rate pL sec 1 0 5 3 0 1 0 5 3 0 Sample Volume pL 10 2 200 3 2 10 Mixing Volume pL 100 5 100 50 5 100 Mixing Speed pL sec 180 25 250 200 25 250 Number of Mixes cycles 2
37. the cytometer for acquisition Exporting a Plate as a Template After you complete setting up a plate you can export it as a template Creating a plate template can save you setup time if you routinely use the same sample setup Plate templates include keywords labels cytometer settings and HTS settings but do not include recorded data 1 Select 96 well U bottom in the Browser and choose File gt Export gt Plate Template or In the Browser select the plate to be exported Select Export gt Plate Template in the shortcut menu The Export Plate Template Wizard appears The bold text at the top of the dialog tells you what to do at each screen See Figure 3 13 on page 77 76 BD High Throughput Sampler User s Guide Figure 3 13 Export Plate Template Wizard Export Plate Template Wizard Select Global Worksheets to include in the Template Export Global Worksheet Oo Global Sheet1 2 Select the global worksheet to include in the template 3 Click Next 4 Select the template type and enter a name The template type is used to group similar templates so they are easy to find At a minimum you need to enter only the type and name when you are exporting a plate template the remaining screens are optional Mi Tip Select the Lock Template checkbox when you want to prevent other users from overwriting your template 5 Click Next and then Finish to view the remaining screens Assigning Keywords Key
38. 0 5 2 0 5 Wash Volume pL 400 200 800 200 200 800 a BD recommends a mixing volume that is one half the available volume See Understanding Volumes on page 25 and Mixing on page 26 NOTICE A mixing volume that is larger than the available volume introduces air bubbles into the sample Shortcut Menu Right click a well in the plate layout to open the shortcut menu The following commands are available in the shortcut menu You can e cut copy and paste wells cytometer settings loader settings and spectral overlap e delete wells and settings e export settings analysis templates and FCS files e import cytometer settings 40 BD High Throughput Sampler User s Guide e apply analysis templates e apply and save cytometer settings e link to a setup e apply compensation controls e apply and save application settings e create an experiment layout Copy Paste Delete Export Import Apply Cytometer Settings viv v v v v Y Apply Compensation kd Application Settings Experiment Layout For additional information about these commands refer to the BD FACSDiva Software Reference Manual Chapter 2 BD FACSDiva Software Overview 41 Renaming Specimens in a Plate To rename specimens in a plate view e Inthe Plate View window click once to select the specimen name in the list of specimens on the plate click the name once more to enter the new name for the specimen then press Ente
39. 113 monthly BD FACSCanto 113 monthly BD LSR I 111 113 surfaces 114 template 105 color indicators sheath fluid 99 well status 35 compensation 81 compensation controls 37 components application window shown 28 HTS shown 16 17 18 consumable part numbers 152 177 controls acquisition 30 conventions user s guide x copy and paste instrument settings 45 wells and specimens 42 coupler sample inspection and service replacing 123 shown 17 18 creating compensation controls 37 customer support xi cytometer connectors 19 21 interface panel 19 21 sample injection tube SIT 21 setting up 59 support bracket shown 20 cytometer settings optimizing 78 80 109 D daily cleaning 104 108 daily maintenance 104 110 data acquiring 82 83 analyzing 86 93 dead volume 25 decontaminating external surfaces fluidics automatically 111 manually 163 defaults acquisition settings HTS settings 40 deleting wells and settings 40 depot repair See repair procedures door sensor cable BD FACSCanto II 167 14 37 18 E error messages acquisition 147 hardware 146 software 149 experiment creating 64 exporting 84 templates 84 Experiment Layout 71 73 accessing from shortcut menu 40 events to record 72 shortcut keys 73 labels 71 exporting experiment templates 84 panel template 85 plate template 76 well settings and files 40 external surfaces decontaminating 167 F fittings hexagonal inspecting 143
40. 2 BD FACSDiva Software Overview 33 34 Plate Toolbar 2a Sa a The Plate toolbar contains the following buttons Z Select a well or group of wells and then click the Add Setup Controls button to add well s to the plate layout for adjusting cytometer settings The wells are for setup only and will not be recorded Ql Select a well or group of wells and then click the Add Specimen Wells button to add a specimen to the plate layout Wells will be run in the order they are selected Select a well that has already been defined and then click the Add Well I button to add another sample or set up a control well The new well is inserted to the right of or below the selected well Click the arrow to the right of the Add Well button to select a horizontal or vertical orientation depending on whether you want to add the new well to the right of or below the selected well ca Select a well and then click the Add Cytometer Settings button to add ov cytometer settings to the sample The Cytometer Settings icon is added to the well in the bottom left corner Select a specimen from th specimen list and then click the Add Cytometer Settings button to add cytometer settings to the selected specimen The Cytometer Settings icon is added to the first well in the top left corner Click the Print button to print the Setup view To save a record of acquisition status for each well print the Setup
41. ACSCanto and BD FACSCanto II Perform this procedure to exchange one sheath fluid with another For example when switching from plate based to tube based acquisition follow these steps to exchange BD FACS sheath solution with surfactant with BD FACSFlow 1 2 3 4 Remove the sample coupler from the SIT Install a cubitainer containing the sheath fluid you want to use Empty the waste tank if necessary Choose Cytometer gt Cleaning Modes gt Prime after Tank Refill to display the Tank Prime window Select FACSFlow then click OK Tank Prime Please select the checkboxes For the tanks that need to be primed Shutdown Solution Cleaning Solution NOTICE It may be necessary to repeat the prime function multiple times to remove air from the sheath lines If air remains in the sheath lines after priming purge air from the sheath filter on the wet cart Refer to your cytometer user s guide for more information on troubleshooting air in the fluid lines 98 BD High Throughput Sampler User s Guide 6 Choose Cytometer gt Sheath Exchange and select the desired sheath exchange neter Fluidics Startup Fluidics Shutdown Cleaning Modes gt Automatic Clean Sheath Exchange FACS Sheath with Surfactant gt FACSFlow FACSFlow gt FACS Sheath with Surfactant Cytometer Details View Configurations CST Performance Tracking LJ ytometer Catalogs Standby
42. Base plate probe assembly plate holder e Sample probe e Pumps e Absorbent pad Monthly Maintenance Procedure Month Completed Initials Date Monthly Cleaning page 111 Surface Inspection and page 114 Cleaning Periodic Maintenance Replacing the Sample page 116 Injection Tubing Replacing the HTS page 119 Sheath Filter Cleaning the Air Filter page 121 Replacing the Sample page 123 Coupler and Tubing Replacing a Pump Syringe page 125 Replacing the Probe page 129 154 BD High Throughput Sampler User s Guide Monthly Maintenance Procedure Month Completed Initials Date Replacing Quick page 130 Connector O Rings Replacing SIT Protector page 130 and O Ring BD LSR II Only Placing HTS Unit into page 132 Long Term Storage Appendix A Consumables and Replacement Parts 155 156 BD High Throughput Sampler User s Guide Appendix B Depot Repair Procedures This appendix contains procedures to perform if your BD High Throughput Sampler HTS needs repair and depot repair service is available in your region For a summary of how the depot repair process works see Depot Repair Overview on page 158 NOTICE Depot repair procedures might be different outside the United States Contact your local BD Biosciences service representative for information for your region The following procedures are cover
43. Chapter 3 Running Samples 83 Stopping the BD High Throughput Sampler The HTS stops automatically when plate acquisition is finished Do the following if you need to stop the HTS in the middle of a run A If you stop the HTS during a run in standard mode the current well will be lost If you stop the HTS in high throughput mode the current and next well will be lost Do not stop acquisition if your sample volume is limited 1 Click Stop Plate __ stoppate or Stop Well s _ sowens in the Acquisition Dashboard The BD High Throughput Sampler finishes the sequence in progress stops and then the following message appears F Sequence Done The Run Stopped NOTICE If you are using the Basic Controls to acquire simply click Stop Acquiring E stop acquiring 2 Click OK to close the dialog Exporting an Experiment as a Template Now that you have completed an experiment with cytometer and HTS settings you can export it as a template Creating Analysis templates for assays that you typically run saves setup time Experiment templates include plates keywords labels worksheet elements and worksheets including all settings such as page breaks cytometer settings and HTS settings but do not include recorded data 1 Select Experiment_001 or whatever you named it in the Browser 84 BD High Throughput Sampler User s Guide 2 Choose File gt Export gt Experiment Template or In the shortcut menu select Exp
44. First wellin group 2 Specimen number Name Concentration E Lymphocytes E Granulocytes Experiment Layout trols Copy b Paste Chapter 3 Running Samples 91 The Batch Analysis dialog appears Batch Analysis V Output To Printer v Statistics View Time 10 v Save as PDF VJ Freeze Biexponential Scales Manual V Use Preferred Global Worksheet Export Filename itistics Batch_Analysis_06022007161404 csv Status 0 6 Select the type of analysis to be done Select Auto to analyze all files with no user intervention Data is displayed in the global worksheet for the amount of time specified in the View Time field in seconds before analysis of the next well begins Make adjustments to your analysis during this pause or let analysis proceed automatically Choose zero only if you want to process the batch without reviewing the data between wells Select Manual to pause the batch after data is loaded for each well Click the Continue button to proceed with analysis of the next well The View Time field is disabled when you select Manual analysis You can print worksheets or export statistics before data for the next well is loaded Output to Printer Print a copy of the analysis for each well Statistics Export statistics to a single CSV file for the batch The resulting file can be opened with a spreadsheet
45. II sample coupler BD FACSCanto and BD FACSCanto II tightening nut sample coupler Make sure the coupler is securely connected to the SIT 13 Tighten the positioning screw under the front of the base plate enclosure 174 BD High Throughput Sampler User s Guide Packing the Unit for Shipping Use the following procedure to pack the inoperable HTS unit prior to shipping NOTICE Pack the unit in the shipping container that contained the HTS replacement unit It should contain the following shipping paperwork decontamination label small I shaped piece of foam plastic bag top piece of foam bottom piece of foam Placing the HTS into Its Shipping Container 1 Make sure the unit was decontaminated according to the procedures in Cleaning the HTS Unit on page 162 Disconnect the sheath and waste lines from the HTS BD FACSCanto II Disconnect the interface communication cable from the HTS Initial and date the decontamination labels stick one on the plate holder and the other on the outside of the shipping carton Enter the initials of the person who decontaminated the cytometer Fill out the Cytometer Return form Place the small I shaped piece of foam between the probe assembly arm and plate holder to secure them Appendix B Depot Repair Procedures 175 7 Slide the HTS unit into the plastic bag A The plastic might make the cytometer slippery and hard to grasp To prevent personal injury or damage to
46. Load data after recording NOTICE Show file identifier GUID in statistics view ensures that the GUID keyword the FCS file s unique identification number appears in the header of statistics views NOTICE Tube specific worksheet and Save analysis after recording through global worksheet options do not apply to plate runs on the HTS Click the New Folder button j in the Browser toolbar Rename the folder Practice To rename any Browser object select it in the Browser and start typing Press Enter to apply the new name With the Practice folder selected click the New Experiment button g in the Browser toolbar A new open experiment is added below the Practice folder in the Browser The experiment contains default cytometer settings and a global worksheet in a Global Worksheets folder Rename the experiment for example with today s date If necessary double click the Inspector button in the Workspace toolbar to show the Experiment Inspector verify that the Use global cytometer settings checkbox is selected see Figure 3 7 on page 66 Chapter 3 Running Samples 65 Figure 3 7 Experiment Inspector Inspector Experiment_001 Experiment Keywords Name Experiment_001 Owner Administrator Modified 1 31 07 1 29 37 PM Log Decades for Plots 4 Log Decades 5 Log Decades Use global cytometer settings checkbox Use global cytometer settings 8 Click on the arrow next to the New
47. Parameter cA R rd zm z linh ese exponential Display sy t Bee 25 989 96 Well U bottom a Esae prr TMT aswel von i TEEN ropo BR 96 Well V bottom pat Biss FSC A 1 000 PerCP Cy5 5 A a28 Shared view 8 a Plot Elements Si 002 D6 Specimen_002 D6 E Piate 96 Well U bottom Osia Epe ESE ee ap Background Color Z Grid Outine Z Tick Marks E za Ea T a Raa Setup Analysis Filter Setup Details Plate Information m a Specimen type Pal Z Acquistion order Z Specimen settings Throughput Mode High Standard E First wellin group 2 Z Specimen number a 7 Well settings ME Acquisition Dashboard Current Activity ee ae E He isi Active Tube Well Threshold Rate Stopping Gate Eve Elapsed Time gea Ust of specimens on the plate C 5e Jo evtis 0 evt 00 00 00 e e Ont Basic Controls w Baw Breo Or Ose ISE Specimen _003 8 a is Oy Plate Controls Brun Brun rx s5 O kJ Loader Settings Sample Flow Rate ul sec 1 0 33 Sample Volume uL E Mixing Volume uL 1 Mixing Speed pL sec EA Number of Mixes ws 2 g p g a E ihs Acquisition Setup PU Stopping Gate ANE Events To R 10000 evt v Stopping Tim Storage Gate AlE v Events To Di 1000 evt Flow Rate
48. Performing Data Analysis 00 0 cece cece nen n eens Maintains Dati ecr 6 srcsaacee ie Ox agiee bebe baa he bee haecn a ol Returning to Tube Based Acquisition 0 0 0 cece eee eee e eee Returning to Tube Based Acquisition on the BD LSR I Returning to Tube Based Acquisition on the BD FACSCanto and BD EACS Canto M sien 3 dos o atte daw de PR at ea a WS oe 8s Shutting Down reia 03 02 ba ee ets alee Pate a a e es SEERA Quitting the Software 2 0 ccc eect nee Chapter 4 Maintenance Daily Maintenance oros renu vice ah ae et A ade Re Ba i Daily Cleaning sactsesecseacig se dined en a eae oe Ea a ee ya eae Cytometer Inspection and Servicing 0 00 cece eee eee eee Monthly Maintenance 0 ccc ete t enn Monthly Cleaning 0 0 0 c cece cece neces Surface Inspection and Cleaning 0 cece ee ee eee eens vi BD High Throughput Sampler User s Guide 64 64 67 74 76 77 78 78 81 82 83 84 84 86 86 90 93 94 94 Periodic Maintenance 0 cece eee eee eens Replacing the Sample Injection Tubing Replacing the HTS Sheath Filter Cleaning the Air Filter 0 0 0 0 cece ee ee eee Replacing the Sample Coupler and Tubing Replacing a Pump Syringe 0 06 Replacing the Probe 0c cece eee eens Replacing Quick Connector O Rings 4 Replacing SIT Protector and O Ring BD LSR II Only P
49. Plate button in the Browser toolbar and select a plate from the drop down list The list contains plates that have been validated with the HTS For details on each plate type see Table 3 1 on page 66 NOTICE The HTS is compatible only with standard depth 96 or 384 well plates BD plates can be ordered from BD Discovery Labware For ordering information visit the BD Discovery Labware website at bdbiosciences com discovery_labware Table 3 1 Plates compatible with the BD High Throughput Sampler Plate Type Well Capacity pL BD Catalog No 384 flat bottom 120 353233 96 flat bottom 300 353915 96 U bottom 300 353910 96 V bottom 340 353263 66 BD High Throughput Sampler User s Guide A Make sure you choose the plate type that corresponds to the plate you will be using BD FACSDiva software cannot verify that the chosen plate matches the plate on the HTS unit If you choose the wrong plate the probe could hit the plate between wells or strike the bottom of a well resulting in damage to the cytometer The selected plate type is added to the experiment If necessary click the Plate button R in the Workspace toolbar to view the Plate window File Edit View Experiment Populations H a Ea P ca FEI T Workspace toolbar Plate button Setting Up the Plate In this section you will add setup controls compensation controls and samples to the plate to run a 6 color experiment As an example you w
50. SCanto Disconnect the power and communication cables from the rear right side of the unit BD FACSCanto II Disconnect the interface communication cable from the cytometer If necessary pull the HTS out slightly to access the cable which is attached to the cytometer at the back left corner of the unit To detach the communication cable unscrew the two connector screws and pull out the cable power cable communication cable BD FACSCanto II Lift the front feet of the HTS unit just over the front edge of the enclosure and tilt the unit at a 45 angle towards you to allow you access to the door sensor cable on the right side of the HTS Unplug the door sensor cable by pulling back on the ferrule Place the cable over the right side of the enclosure carrier Carefully lift the unit and place it on the benchtop next to the cytometer BD LSR II and BD FACSCanto only Remove the absorbent pad from the drip reservoir and discard it as biohazardous waste Figure B 2 Appendix B Depot Repair Procedures 161 Figure B 2 Drip reservoir containing absorbent pad BD LSR II and BD FACSCanto only absorbent pad 15 Place the sample tubing and attached sample coupler inside the drip reservoir Cleaning the HTS Unit You must decontaminate both the internal fluidics and external surfaces of your HTS unit before it is repaired If you were able to run a monthly clean before you removed the HTS unit the internal fluidics are d
51. The following components are available in the Analysis view Plate Layout The plate layout in the default Analysis view displays the acquisition status of each well See Table 2 2 on page 36 for status indicators If keywords were assigned to the plate clicking on a keyword in the Keywords list toggles the plate layout to show the Keywords Analysis view See Figure 2 3 Each keyword is shown as a different color If a keyword was assigned a range of values a value is displayed in each well Different values of the same keyword are shown in various shades of the keyword color If a well has no value assigned the well is white To revert back to the default Analysis view click the keyword in the Keywords list Figure 2 3 Plate layout showing Keyword Analysis view Add Keyword button E Plate 96 Well U bottom Setup Analysis Legend Keywords gt A ec 5 z G m a Specimen type pE First well in group W Specimen number Nene Suffix Fconcentraton E Lymphocytes Oo aie Keyword list List of specimens on the plate E Setup Controls_001 E Compensation Controls 3 Specimen_001 4 Specimen_002 Chapter 2 BD FACSDiva Software Overview 49 Add Keyword Button Click the Add Keywords button 4j to bring up the Add Keywords dialog box Figure 2 4 The Available Keywords list displays all keywords that were used in the
52. acing the Probe on page 129 A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Treat the sample probe and tubing as biohazardous waste and dispose of them according to local regulations Figure B 3 Removing the sample probe and tubing sample probe sample probe tubing 2 Pipette a 10 bleach solution into the injection port wash station Using a disposable pipette fill the injection port wash station completely being careful not to overfill 3 Connect the line declogger tool to the waste connector on the back of the unit Figure B 4 Attaching the declogger tool F 164 BD High Throughput Sampler User s Guide Slowly pull back the plunger of the declogger tool until all of the bleach solution is drawn into the barrel Disconnect the declogger tool and discard the bleach solution according to local regulations Repeat step 2 Reattach the declogger tool and slowly remove about half of the bleach solution allow the remaining bleach to sit for 30 minutes Keep the declogger tool attached to the waste port during the 30 minutes Use the declogger tool to remove the rest of the bleach solution Disconnect the declogger tool and discard the bleach solution according to local regulations Rinsing the Fluidics 1 n GO UU A Pipette DI water into the injection port wash station Using a disposable pipette fill the injection port wash station comp
53. age 114 e Unscheduled Maintenance on page 136 103 Daily Maintenance When running the HTS daily perform the following at the end of every day or shift e Daily Cleaning see the following section e Cytometer Inspection and Servicing see page 109 NOTICE BD LSR II When running samples containing acridine orange or propidium iodide run Daily Clean twice First run 70 isopropyl alcohol in wells A1 A4 and BD FACSClean or a 10 bleach solution in wells B1 B4 Second run BD FACSRinse solution in wells A1 A4 followed by DI water in wells B1 B4 Daily Cleaning During the daily clean procedure the cytometer samples cleaning solution and then DI water from predefined wells and performs a sequence of mixing aspiration and rinsing Software prompts guide you through the cleaning sequence Perform the cleaning procedure at the end of every day or shift while the cytometer is in plate based mode Allow 15 minutes to complete this procedure Materials Needed e BD FACSClean or a fresh 10 bleach solution 1 part bleach in 9 parts DI water e BD FACSRinse solution e DI water 104 BD High Throughput Sampler User s Guide Running Daily Cleaning 1 Choose HTS gt Clean The Plate Templates dialog appears Figure 4 1 on page 105 Figure 4 1 Plate Templates dialog Plate Templates a Date Name Daily Clean 96 well U bottom 96 Well U bottom 1 25 07 4 38 PM of cleaning solution Daily Cle
54. aling washers in each port If you over tighten the fitting the sealing washers can be compressed resulting in a blocked port Tighten the fitting until it contacts the sealing washer and then turn the fitting an additional 1 6 to 1 4 turn If leaking is observed tighten the fitting no more than an additional 1 8 turn 4 Reinstall the HTS unit on the cytometer For detailed instructions see Installing the HTS Unit on page 170 124 BD High Throughput Sampler User s Guide Replacing a Pump Syringe Glass syringes are used in the primary and secondary pumps in the HTS unit Replace a syringe once a year or whenever leaks occur or pumping accuracy is suspect To begin this procedure the cytometer and software must be running A All cytometer surfaces and hardware that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when changing the syringe Wear suitable protective clothing eyewear and gloves 1 Before replacing the pump syringe perform monthly cleaning See Monthly Cleaning on page 111 2 BD LSR II Place the cytometer in Run mode 3 Remove the liquid from the syringe by performing steps 2 through 11 in Placing HTS Unit into Long Term Storage on page 132 4 Turn off the HTS power switch BD LSR II and BD FACSCanto or turn off the cytometer BD FACSCanto II Keep the software running on the workstation 5 If you are replacing e the primary pump syringe located
55. an 96 well U bottom 2 28 06 2 02 PM eee of rinse solution Name Daily Clean 96 well U bottom 2 Select the Daily Clean 96 well U bottom template if not already selected If you do not have a U bottom plate for cleaning you can set up your own cleaning template 3 Click OK e The Plate Interface changes to show the Daily Clean Protocol view Figure 4 2 on page 106 Chapter 4 Maintenance 105 Figure 4 2 Daily Clean Protocol view E Plate Daily Clean 96 well U bottom Filter Setup Details Plate Information on Specimen type Fs Acquisition order i Specimen settings Throughput Mode High Standard First well in group bz V Specimen number V Well settings ae B its Plate Status Injecting Sample For Pump1 List of specimens on the plate F Clean A IA Rinse 2 1 J 2 Loader Settings ICS i Sample Flow Rate uL sec Sample Volume UL Mixing Volume uL Mixing Speed uL sec Number of Mixes Wash Volume UL 400 eF e The following message appears Figure 4 3 Cleaning confirmation message Install the prepared cleaning tray on the HTS Ca e NOTICE Do not click OK at this time 4 Fill the wells of a 96 well plate according to the following table Wells Solution Volume uL A1 A4 BD FACSClean 200 B1 B4 DI water 200
56. and pasting 42 create Experiment Layout cytometer settings in 40 deleting 40 displaying keywords 86 exporting settings and files loader settingsin 40 perform panel analysis 40 spectral overlap 40 volume 25 windows Acquisition Dashboard 30 Browser 29 Inspector 31 Plate 31 worksheet global 65 normal 76 setting up 74 Index 183 184 BD High Throughput Sampler User s Guide
57. ansmit potentially fatal disease Use universal precautions when changing O rings Wear suitable protective clothing and gloves 130 BD High Throughput Sampler User s Guide 1 Remove the safety cover from the HTS 2 Detach the sample coupler from the cytometer SIT 3 Remove the SIT protector Figure 4 16 Unscrew the tube retainer and slide the SIT protector straight down A To avoid bending the SIT do not slide the SIT protector at an angle Figure 4 16 Removing the SIT protector tube retainer SIT protector 4 Slide the tube retainer down the metal tubing to find the O ring O ring If the O ring is stuck inside the retainer push the retainer to the top of the tubing and work it down at an angle until the O ring is free 5 Remove the O ring Chapter 4 Maintenance 131 6 Install a new O ring and slide it to about the middle of the tube O rings are included in the accessory kit 7 Slide the SIT protector straight onto the SIT A To avoid bending the SIT do not slide the SIT protector at an angle 8 While holding the tube gently push up on the retainer screw and screw it on to secure it 9 Reattach the sample coupler to the cytometer SIT Slide the sample coupler onto the SIT until you reach a hard stop Make sure the sample coupler tubing is not kinked or twisted Hold the coupler with one hand while you tighten the top nut with the other hand Note that there should be a gap between the tightening nu
58. application such as Microsoft Excel Depending on the auto export format selected in User Preferences each well adds a new row or column of results to the file For each population in the statistics view the software adds parameter statistics in the order in which they appear in the view A new header row is added if you add or delete statistics or parameters during batch analysis You cannot add remove or edit statistics views while the batch is running 92 BD High Throughput Sampler User s Guide Save as PDF the Add Report to PDF and View PDF checkboxes become active If you keep Add Report to PDF selected the Batch Analysis Report is added at the top of the worksheet s PDF file If you keep View PDF selected the PDF is automatically displayed at the completion of the batch analysis Specify whether to let biexponential scales fluctuate if applicable When the Freeze Biexponential Scales checkbox is selected biexponential scaling does not change during batch analysis All data is processed using scales from the tube where the current tube pointer is set For information on biexponential scales refer to the BD FACSDiva Software Reference Guide Preferred global worksheets can be used with wells and specimens For information on using preferred global worksheets refer to the BD FACSDiva Software Reference Manual NOTICE Choose zero only if you want to process the batch without reviewing data between wells 7 Click Star
59. ase Treat the secondary pump valve tubing as biohazardous waste and dispose of it according to local regulations 8 Detach the sample coupler tubing from the secondary pump valve and discard the tubing A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Treat the tubing and sample coupler as biohazardous waste and dispose of them according to local regulations 9 Cover the open ports with parafilm or tape Decontaminating External Surfaces Materials Needed e 10 bleach solution e DI water e paper towels Cleaning the Surface of the HTS Unit A To prevent cytometer damage do not pour or squirt bleach solution directly onto the cytometer 1 Saturate some paper towels with a 10 bleach solution 2 Move the plate holder to the far left wipe down the surface of the stage that was under the plate holder using the saturated paper towels see Figure B 7 on page 168 Appendix B Depot Repair Procedures 167 Figure B 7 HTS unit top view 3 Repeat step 2 moving the plate holder to the far right front position and then back position 4 Clean the remaining external surfaces of the cytometer using paper towels saturated with a 10 bleach solution Make sure all surfaces are clean 5 Dispose of the paper towels used for cleaning as biohazardous waste 6 Rinse all surfaces by repeating steps 1 through 5 substituting DI water for the bleach solution 7 Allow the
60. ate window Use this window also to select the throughput mode and adjust loader settings E Plate 96 Well U bottom Setup Analysis Filter Setup Details Plate Information o rac 5 A a0 Specimen type V Acquisition order 3 Specimen settings Throughput Mode High Standard v pE First wellin group b Specimen number a 7 Well settings Plate Status iinader Stohie List of specimens on the plate 1 Setup Controls_001 EE Compensation Controls 3 Specimen_001 Loader Settings Sample Flow Rate yL sec 10 Sample Volume uL 10 BB Mixing Volume uL 100 Sf Mixing Speed uL sec i g Speed L sec 180 Sf Number of Mixes 2 Wash Volume uL 400 Bp Chapter 2 BD FACSDiva Software Overview 31 Plate Window Most of the features for running plate based experiments on the HTS are located in the Plate window Figure 2 2 on page 32 The tabs at the top of the Plate window represent views that organize the workflow for the plate The Setup view is for setting up the experiment and the cytometer adjusting cytometer settings and acquiring and analyzing samples from a plate the Analysis view is for assigning keywords to wells and analyzing data after a run The Plate window is available when an experiment is open and you double click the plate in the Browser
61. ating the type of specimens in the plate and allows you to filter hide or show acquisition order specimen number specimen settings and or well settings Fitter Setup Details m Specimen type s Acquisition order iSd J7 Specimen settings E First well ingroup b JV Specimen number a IV Well settings Click the checkbox next to acquisition order specimen number specimen settings and or well settings to clear the checkmark and hide the symbol in the plate layout Specimen type Indicates the type of control or sample assigned to a given a well The pink square represents a setup control the purple square a compensation control and the blue circle a specimen First well in group A dark blue square appears for the specimen number in the upper right corner of the first well for each specimen Acquisition order filter The order sequence number in which each well will be acquired appears with a green background in the bottom right corner of the well z Specimen number The specimen number appears in the upper right corner E Each well belonging to the same specimen will have the same specimen number 5 Specimen settings When cytometer settings are added to a specimen the ra cytometer settings icon appears in the upper left corner of the well Well settings When cytometer settings are added to a well the cytometer i settings icon appears in the lower left corner of the well Chapter
62. bdbiosciences com Part No 642224 Rev A June 2007 BD High Throughput Sampler User s Guide for the BD LSR II BD FACSCanto BD FACSCanto II M BD Biosciences 2350 Qume Drive San Jose CA 95131 1807 USA Tel 877 232 8995 Fax 800 325 9637 facservice bd com Asia Pacific Tel 65 6 861 0633 Fax 65 6 860 1590 Europe Tel 32 2 400 98 95 Fax 32 2 401 70 94 help biosciences europe bd com Brazil Tel 55 11 5185 9995 Fax 55 11 5185 9895 Japan Nippon Becton Dickinson Company Ltd Toll Free 0120 8555 90 Tel 81 24 593 5405 Fax 81 24 593 5761 Canada Toll Free 888 259 0187 Tel 905 542 8028 Fax 888 229 9918 canada bd com Mexico Toll Free 01 800 236 2543 Tel 52 55 5999 8296 Fax 52 55 5999 8288 2007 Becton Dickinson and Company All rights reserved No part of this publication may be reproduced transmitted transcribed stored in retrieval systems or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without prior written permission from BD Biosciences The information in this guide is subject to change without notice BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments Although this guide has been prepared with every precaution to ensure accuracy BD Biosciences assumes no liability for any e
63. cytometer to dry thoroughly 168 BD High Throughput Sampler User s Guide Unpacking the Replacement Unit AN A HTS unit without a cover weighs approximately 22 lb To prevent personal injury or damage to the cytometer follow the guidelines on page 169 to remove the replacement HTS unit from its shipping container NOTICE If you are sending back an existing HTS for depot repair save all shipping materials and ship the existing unit in this new container It contains paperwork for shipping and two labels verifying that the cytometer you are returning was decontaminated Before you proceed with unpacking e Make sure you have adequate space onthe floor near the cytometer to place the shipping container onthe table or bench near the cytometer to place the new HTS unit e Make sure you read through these instructions before attempting to unpack the new cytometer are ready to install the new cytometer See Installing the HTS Unit on page 170 Lifting Heavy Objects A To avoid personal injury or damage to the cytometer follow these guidelines for lifting heavy objects from the floor 1 Stand in front of the object and then sit on your heels squat 2 Place your hands under the base of the object ensure the load is balanced and bring the object as close to your lap as possible Appendix B Depot Repair Procedures 169 3 While keeping your spine in a natural position and your head up lift the object using the fo
64. derneath the catch tray tongue and sample coupler slot Push the tray in making sure the coupler line is not crimped or kinked Push in the unit and tighten the positioning screw holding the HTS to the cytometer support bracket Cleaning the Air Filter The air filter element is located within a filter retainer and fan guard attached to the base plate of the HTS unit To clean or replace the air filter follow these steps 1 Remove the HTS unit from the cytometer For detailed instructions see Removing the HTS Unit on page 159 Chapter 4 Maintenance 121 2 Place the HTS unit on its side NOTICE To avoid scratching the finish handle the HTS with care Do not drag the unit on the laboratory bench 3 Remove the filter retainer The filter retainer is located within the air filter element in the base plate of the unit Example of filter for BD LSR Il and BD FACSCanto filter retainer e Place your middle and index fingers of each hand on the opposite edges of the filter retainer place your thumbs on the fan guard through the retainer apertures e Support the fan guard with your thumbs as you pull out the retainer with your fingers NOTICE To prevent the HTS unit from tipping use your thumbs to hold the unit in place while pulling 122 BD High Throughput Sampler User s Guide 4 Remove the filter and clean or replace it as needed filter filter retainer To clean the filter rinse it well with water Dry
65. e fluid control panel on the cytometer itself Setting Up for Plate Based Acquisition This section describes how to set up the cytometer for plate based acquisition A Any cytometer surface that comes in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling cytometer hardware Wear suitable protective clothing and gloves Setting Up for Plate Based Acquisition on the BD FACSCanto and BD FACSCanto II When switching from tube to plate mode perform a sheath fluid exchange then attach the sample coupler to the SIT 1 Turn on the cytometer and the HTS For the BD FACSCanto turn on the cytometer followed by the HTS For the BD FACSCanto II the HTS turns on automatically when the cytometer is powered on 2 Remove open the HTS safety cover BD FACSCanto Tilt the front of the cover up then slide the cover forward to remove it BD FACSCanto II Slide open the covers Slide the front cover to the left slide the side cover to the back 3 Perform a sheath fluid exchange Refer to Exchanging the Sheath Fluid on a BD FACSCanto and BD FACSCanto II on page 98 Chapter 3 Running Samples 59 60 4 Attach the HTS sample coupler to the cytometer SIT Move the aspirator arm to the left Then slide the sample coupler onto the SIT until you reach a hard stop Make sure the sample coupler tubing is not kinked or twisted Hold the coupler with one hand while you tighten the t
66. econtaminated skip to Decontaminating External Surfaces on page 167 If you were not able to run a monthly clean proceed with the following section A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when cleaning the cytometer Wear suitable protective clothing eyewear and gloves 162 BD High Throughput Sampler User s Guide A At prevent shock verify that the cytometer is turned off and the power cable is detached before you start cleaning Use a 10 bleach solution to decontaminate the internal fluidics and external surfaces of the HTS unit A To ensure that the 10 bleach solution retains its full germicidal effect prepare a fresh solution daily 1 Pour 45 mL of DI water into a 100 mL beaker 2 Carefully add 5 mL of undiluted bleach to the same beaker 3 Gently swirl the solution to mix Decontaminating the Fluidics Manually Materials Needed e household bleach e DI water e disposable plastic pipettes e line declogger PN 343583 from the accessories kit for manual fluidics decontamination only Cleaning the Fluidics Perform this procedure to decontaminate the internal fluidics of a non functional HTS unit 1 Remove the sample probe and tubing that connects the probe to the primary pump discard the probe and tubing Figure B 3 on page 164 Appendix B Depot Repair Procedures 163 For details on removing the probe see Repl
67. ed in this chapter Depot Repair Overview on page 158 Removing the HTS Unit on page 159 Cleaning the HTS Unit on page 162 Unpacking the Replacement Unit on page 169 Installing the HTS Unit on page 170 Packing the Unit for Shipping on page 175 157 Depot Repair Overview BD HTS has problems Consultuser s guide Troubleshooting section ns enon No Call BD Technical Support 1 877 232 8995 Follow Technical Support Representative instructions Problem 5 No The Technical Support Representative will schedule HTS replacement deliveryto your laboratory Record the notification number for tracking the retum of your maltunctioning HTS Unit must be retumed within S business days Referto the Unpacking Instructions in the user s gude Open the box Unpack the HTS replacement Referto the Decontamination Instructions in the user s guide Decontaminate the malfunctioning HT per Monthly Cleaning procedure or Manual Decontamination procedure accordingly Fill in and attach Decontarinated label on the plate holder Locate the hstrument Retum Form inside the HTS replacement box Complete and sign the hstrurment Retum Form Referto the HTS Customer Installation Removal Instructions in the user s guide 158 BD High Throughput Sampler User s Guide Uninstall the maltunctioning HTS hstall HTS replacement and execute performance venication Re
68. ee Exchanging the Sheath Fluid on a BD LSR II on page 96 or Exchanging the Sheath Fluid on a BD FACSCanto and BD FACSCanto II on page 98 To prevent bubble formation in the flow cell use only BD FACS sheath solution with surfactant BD Catalog No 336524 US or 336911 Europe when acquiring samples with the HTS This sheath solution is intended for research use only and should be used only with the HTS option It should not be used for sorting or in vitro diagnostic IVD applications Refer to the product insert for more information Add 500 pL of Sigma Antifoam A Concentrate to the waste tank to prevent foaming of potentially biohazardous waste up around the cap Mix the Antifoam Concentrate in distilled water to force it into solution before adding it to the waste tank Add the Antifoam Concentrate in addition to bleach Do not allow the system to run dry as this could damage the HTS pumps Turn on the HTS NOTICE The HTS turns on automatically for the BD FACSCanto II Ensure the sample coupler is installed See details under Setting Up for Plate Based Acquisition on page 59 BD High Throughput Sampler User s Guide Start up the computer launch BD FACSDiva software P gH Double click the shortcut on the desktop BD FACSDiva Software Verify that no other software applications are running before you start acquisition BD FACSDiva software performance time can be severely affected if multiple applications are runnin
69. er 336218 1 Absorbent pad BD LSR II and BD FACSCanto 343502 1 Injection tubing to secondary pump 335451 2 152 BD High Throughput Sampler User s Guide Table A 2 Other replacement parts and reagents Item Part No SIT protector assembly BD LSR II 335345 BD LSR II sheath line assembly 336076 BD LSR II waste line assembly 336077 BD FACSCanto sheath line assembly 7000949 BD FACSCanto waste line assembly 7000950 BD FACSCanto II sheath line assembly 700097807 BD FACSCanto II waste line assembly 700097707 Line declogger tool 343583 Air filter retainer 336474 5 16 in open ended wrench 337016 BD FACS sheath solution with surfactant 336524 BD FACSClean 340345 BD FACSRinse solution 340346 Anti foam concentrate 25 g bottle 334887 Cleaning stylus 343648 Purging assembly line BD LSR II 340088 HTS safety cover BD LSR II and BD FACSCanto 640404 2 ft serial cable BD FACSCanto 640737 HTS catch tray BD FACSCanto 640576 HTS power communication cable BD FACSCanto II 344368 Appendix A Consumables and Replacement Parts 153 Maintenance Log Use the following log to keep track of maintenance procedures on your HTS unit You can photocopy the log and keep it next to the cytometer or use it as a guide to design your own Daily Maintenance Procedure Daily Cleaning page 104 Cytometer Inspection and Servicing page 109 e Sampler coupler e
70. er user s guide If additional assistance is required contact your local BD Biosciences service representative See Technical Assistance on page xi Troubleshooting suggestions in this chapter are grouped under the following headings e HTS Troubleshooting on page 146 e Acquisition Troubleshooting on page 147 e BD FACSDiva Troubleshooting on page 149 145 HTS Troubleshooting Observation Bubbles in sample wells Possible Causes Mixing volume too great Recommended Solutions Reduce the mixing volume See Understanding Volumes on page 25 and Mixing on page 26 for recommendations Too many mixes Reduce the number of mixes Insufficient sample volume Increase sample volume Leaking around sample coupler or cytometer SIT Coupler or tube retainer loose or worn Check the fittings and tighten them as needed Leaking around pump syringes Fitting loose Tighten the fitting and replace it if necessary See Inspecting Thumbscrew Fittings on page 142 BD LSR II DCM system running when tube arm to side Acquisition switch in Tube mode Switch to Plate mode Bubbles in waste tank No antifoam concentrate being used Add 500 pL Sigma Antifoam A Concentrate to the waste tank Fluid accumulating around the injection port wash station Sample probe assembly is bent Replace sample probe assembly Clogged wash station e Add water to the injection port wa
71. erform batch analysis you must view data using a global worksheet For an example of setting up and performing batch analysis see Performing Batch Analysis on page 90 Chapter 2 BD FACSDiva Software Overview 53 54 BD High Throughput Sampler User s Guide 3 Running Samples This chapter describes how to set up for plate based acquisition and acquire samples using the BD High Throughput Sampler For general information about the software see Chapter 2 Mi Tip Ifyou are running the system for the first time BD Biosciences recommends that you practice running a sample plate using BD Calibrite beads or a similar control sample The following topics are covered in this chapter Starting Up on page 56 Creating Experiments on page 64 Preparing for Acquisition on page 78 Performing Compensation on page 81 Acquiring Data on page 82 Analyzing Data on page 86 Maintaining Data on page 93 Returning to Tube Based Acquisition on page 94 55 Starting Up 56 This section describes how to start up the cytometer and software Notice that some steps apply only to a specific cytometer s 1 Start up the flow cytometer as described in the appropriate cytometer manual Make sure to refill the sheath container and empty the waste container If a full waste container is detected at the start of or during a run the run will be stopped If necessary perform a sheath fluid exchange to use the appropriate sheath solution S
72. essary parameters in the Parameters tab of the Cytometer Inspector 68 BD High Throughput Sampler User s Guide The parameters should match those shown in the following figure Inspector Cytometer Settings Cytometer Settings Parameters Threshold Ratio Compensation Parameter Voltage FSC 250 55C 300 FITC PE 500 O vs PerCP Cy5 5 PE Cy APC APC Cy oOoloooldldlo Ooldloldlol SSS 8 8 8 0 0 KS 3 KS 1 C3 ES KS he 4 Click and drag to select wells A1 and A2 in the plate layout 5 Click the Add Setup Controls button S in the Plate toolbar to add setup control wells to the experiment These two wells will be used for unstained control to set threshold and PMTs Mi Tip Designate at least two wells as setup controls to make sure that you do not run out of sample while optimizing cytometer settings 6 Select well B1 and choose Experiment gt Compensation Setup gt Create Compensation Controls 7 Verify that the options in Figure 3 8 on page 70 are selected Chapter 3 Running Samples 69 Figure 3 8 Create Compensation Controls dialog Plate button Create Compensation Controls O Plate Include unstained Include separate unstained control tube well control checkbox Fluorophore FITC Generic PE Generic e PerCP Cy5 5 Generic e PE Cy
73. et e Store the base plate with the HTS unit Chapter 4 Maintenance 135 Placing the HTS for BD FACSCanto or BD FACSCanto II in Long Term Storage 1 Ensure the sample coupler is installed and the HTS is turned on 2 Perform a fluidic shutdown See Fluidics Shutdown on page 100 for details 3 If required remove the HTS from the cytometer See Removing the HTS Unit on page 159 Unscheduled Maintenance Perform the following procedures when necessary or when you are directed to do so by a BD Biosciences service representative e Homing the Sample Probe on this page e Priming the HTS on page 138 e Performing a Motion Test on page 138 e Verifying the Sample Probe Position on page 140 e Inspecting Thumbscrew Fittings on page 142 e Inspecting Hexagonal Fittings on page 143 e Declogging the SIT on page 143 Homing the Sample Probe During normal operation the sample probe goes through a homing sequence during initialization Perform the following procedure to manually send the probe to the home position or when directed by a BD Biosciences service representative during troubleshooting 136 BD High Throughput Sampler User s Guide 1 BD LSR II Place the cytometer in Run mode and ensure that the sample coupler is installed The software automatically primes the HTS pumps during a homing operation To prevent pressure from building in the flow cell the cytometer must be in Run mode before the homing sequence begins If
74. etup Stopping Gate i allEvents y Events To Record 10000evt v Stopping Time s Storage Gate _ ll Events v Events To Display 1000evt _ Flow Rate Acquisition Status Processed Events Electronic Abort Rate Threshold Count Electronic Abort Count You can expand show and contract hide the Acquisition Dashboard Hide Plate Control Hide Acquisition Setup Hide Acquisition Status Hide All To show or hide the Plate Controls Acquisition Setup or Acquisition Status sections of the Acquisition Dashboard right click the Acquisition Dashboard in any blank area except for Basic controls to display the shortcut menu You can resize the Acquisition Dashboard using standard Windows methods 30 BD High Throughput Sampler User s Guide KG Use the Inspector to view or modify the attributes of a single object or set of objects on the Inspector A wet HTS Labels Aca Keywords worksheet or plate or in the Browser The Sample Flow Rate uiL sec Lom contents of the Inspector change depending on Sample Volume uL ga what is selected in the workspace BESET so j The Inspector can be used to view HTS Loader Mixing Speed ul sec 200 settings To modify these settings use the Loader Number of Mixes oi Settings in the Plate window see Loader Settings Wash Volume uL 20 EA on page 38 a Set up acquire and analyze plate based experiments in the Pl
75. ferto the Packing Instructionsin the user s guide Use the packing material from the HTS replacement box to pack the maltunctioning HTS Place the hstrurent Retum Form on top ofthe upper foam Close the box with tape Attach a Decontarinated label onthe front ofthe 2 day shipping box Attach the UPS ARS UPS label overthe senice previous shipping label Vitite on the box the notification number provided by your Technical Support Representative Present the carrier with the box within the 5 day allotted ime Removing the HTS Unit Follow this procedure to detach your HTS unit from the cytometer to perform maintenance specified in this user s guide or before sending it in to BD Biosciences for depot repair A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling cytometer parts Wear suitable protective clothing eyewear and gloves NOTICE If you are able to run the system decontaminate the HTS before you remove it by performing a monthly cleaning procedure See Monthly Maintenance on page 111 If you cannot run the system decontaminate it after removal as described in Cleaning the HTS Unit on page 162 1 2 3 4 BD LSR II and BD FACSCanto Switch off the HTS power Shut down the cytometer Remove or open the HTS safety cover Detach the sample coupler from the cytometer SIT e Unscrew the top nut
76. filter and waste orange Door sensor cable connector cable detects if safety door is open Injection port wash station provides interface for sample injection and probe washing Sample coupler connector between HTS unit to cytometer SIT Plate holder moves left to right and front to back to position plate so the probe can pick up sample Secondary pump and valve delivers sample to flow cell in high throughput mode Overflow reservoir collects potential overflow from the injection port wash station or drips from the cytometer SIT CRORC EC COROT CES 18 BD High Throughput Sampler User s Guide Cytometer Connections Cytometer Interface Panel Sheath and waste travel between the cytometer and the HTS unit via connectors in the cytometer interface panel see Figure 1 5 Figure 1 6 and Figure 1 7 The BD LSR I interface panel also includes an acquisition mode switch which controls pressure at the cytometer sample injection tube SIT e In Tube mode W the droplet containment module DCM vacuum functions normally the vacuum is on when the arm is positioned to the side and off when the arm is centered Backdripping from the sample injection tube is contained when the DCM sleeve is installed e In Plate mode 32 the DCM vacuum on the cytometer does not function Drip containment is provided by the absorbent pad in back of the HTS unit Figure 1 3 on page 17 A BD LSR II To keep your cytometer
77. free of drips from potentially biohazardous samples always switch the cytometer to Tube mode and install the DCM sleeve when you are not acquiring samples using the HTS option Note that if backdripping does occur drips are contained by the absorbent pad in back of the HTS unit Chapter 1 Introduction 19 Figure 1 5 Cytometer interface panel BD LSR II Tube Plate acquisition mode switch sheath connector waste connector waste tubing sheath tubing cytometer support bracket power cable communication cable base plate HTS positioning screw Figure 1 6 Cytometer interface panel BD FACSCanto communication cable waste tubing sheath tubing power cable base plate 20 BD High Throughput Sampler User s Guide Figure 1 7 Cytometer interface panel BD FACSCanto II interface sheath connector communication connector NOTE The white air waste connector is used for the connector pneumatic tube loader door sensor cable enclosure Sample Coupler A sample coupler connects the injection port tubing on the HTS unit to the cytometer sample injection tube SIT Figure 1 8 on page 22 For the BD LSR II the droplet containment module DCM sleeve is replaced by the SIT protector The SIT protector is a modified sleeve that prevents the sample injection tube from bending during installation of the HTS sample coupler For the BD FACSCanto and BD FACSCanto II the sample coupler is i
78. g at the same time At the Log In dialog choose your user name enter your password and click OK RAOBD i S User Name amp administrator S Password R OK Quit For instructions on creating a user name and password refer to the BD FACSDiva Software Reference Manual Verify that the software connects to the cytometer If the software does not connect choose Cytometer gt Connect View connectivity status at the bottom of the Cytometer window Figure 3 1 on page 58 Chapter 3 Running Samples 57 Figure 3 1 Cytometer window Cytometer FACSCantoll 1 x tuS Parameters Threshold Laser Compensation Ratio Time Event connectivity status The system is ready l l m E o 7 BD FACSCanto and BD FACSCanto II Perform a fluidics startup Choose Cytometer gt Fluidics Startup The following message appears Fj Confirm Fluidics Startup can take up to 10 min Click OK to initiate Click Cancel to abort Cancel Click OK to begin fluidics startup When fluidics startup is complete the following message appears Startup Status i Fluidics startup complete The system is ready Click OK 58 BD High Throughput Sampler User s Guide 8 BD LSR II Place the cytometer in Run mode then choose HTS gt Prime to prime the HTS unit When the priming is complete click OK in the dialog NOTICE To prime the cytometer press the Prime button on th
79. he door sensor cable that is attached to the cytometer over the right side of the enclosure BD LSR II and BD FACSCanto Place the HTS on the base plate carrier BD FACSCanto II Bring the unit close to the cytometer while you sit in a chair with the unit in your lap Make sure the unit support legs fit into the bracket grooves With the legs in the groove slide the unit as far forward as possible BD LSR II and BD FACSCanto Connect the power and communications cables to their respective ports on the rear right side of the unit see Figure B 8 on page 171 Figure B 8 Connecting the power and communication cables example of BD LSR II power cable communication cable BD FACSCanto II Tilt the unit at a 45 angle towards you to allow you access to the door sensor connector on the right side of the unit Plug the door sensor connector into the HTS unit matching the white arrow on the plug with the arrow on the connector see Figure B 9 Appendix B Depot Repair Procedures 171 door sensor cable Match the arrow on the plug with the arrow on the HTS connector 6 BD FACSCanto II If necessary slide the unit in until you can connect the interface communication cable to the cytometer see Figure B 10 on page 173 Tighten the two connector screws after the communication cable is attached 7 BD FACSCanto II Install the overflow reservoir with the lip facing towards you and place the overpressure tubing int
80. ic force will secure the sample injection port wash station in place 9 Reconnect the secondary pump valve tubing F to the secondary pump by tightening the hex screw B 10 Reinstall the HTS unit Follow the instructions for Installing the HTS Unit on page 170 118 BD High Throughput Sampler User s Guide Replacing the HTS Sheath Filter The HTS sheath filter is integrated in the fluid line that supplies sheath fluid to the HTS unit Figure 4 9 on page 120 Replace the filter every 6 months A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling cytometer components Wear suitable protective clothing eyewear and gloves 1 Loosen the positioning screw holding the HTS to the cytometer support bracket Figure 4 8 Figure 4 8 Loosening the positioning screw example shown for BD LSR II support bracket positioning screw 2 Slide the HTS unit towards you by doing the following e Slightly lift the HTS unit to clear the screw e Being careful not to strain the sheath tubing slide the unit towards you until you can access the filter Chapter 4 Maintenance 119 Figure 4 9 Sheath line and filter example shown for BD LSR II sheath line filter 3 BD FACSCanto Remove the catch tray Pull the HTS towards you slightly to allow room for removing the catch tray 4 Label the sheath line connectors To keep track
81. igure 1 2 HTS unit front view example of HTS Plate holder moves left to right and front to back to position plate so the probe can pick up sample Primary pump and valve enables mixing and aspiration of sample delivers sample to flow cell in standard mode OFC RC Power switch and LED indicator for HTS unit 16 BD High Throughput Sampler User s Guide Figure 1 3 HTS unit for BD LSR II and BD FACSCanto rear view GOTON ONO Probe assembly moves front to back and up and down to transfer sample between plate holder and injection port wash station Fluidics tubing sheath clear and waste orange Sheath filter filters incoming sheath fluid to HTS unit Injection port wash station provides interface for sample injection and probe washing Plate holder moves left to right and front to back to position plate so the probe can pick up sample Secondary pump and valve delivers sample to flow cell in high throughput mode Absorbent pad collects potential overflow from the injection port wash station or drips from the cytometer sample injection tube SIT Sample coupler connector between HTS unit to cytometer SIT Chapter 1 Introduction 17 Figure 1 4 HTS unit for BD FACSCanto II rear view Probe assembly moves front to back and up and down to transfer sample between plate holder and injection port wash station Fluidics tubing sheath clear with in line
82. ill be recording and analyzing human peripheral blood stained with the following reagents CD14 FITC CD16 CD56 PE CD 8 PerCP Cy 5 5 CD19 PE Cy 7 CD3 APC CD4 APC Cy7 You will need seven compensation control wells to accommodate the unstained control and the six fluorochromes you are using in your experiment 1 If necessary double click the Inspector button in the Workspace toolbar to view the Plate Inspector select High Throughput Mode Inspector 96 Well U bottom Plate Name 96 Well U bottom Throughput Mode High Standard Plate Type Chapter 3 Running Samples 67 Or select the throughput mode in the upper right corner of the Plate window s Setup view Plate Information Throughput Mode High C Standard Plate Status Loader Status NOTICE Setup and compensation control wells are always acquired in standard mode even if high throughput mode is selected in the Plate Inspector Sample wells are acquired using the throughput mode selected in the Plate Inspector unless you acquire individual wells manually See Running Wells Manually on page 47 for more information 2 Inthe Browser click on the Experiment global Cytometer Settings P2 Browser Blank Experiment with Ge lt Fit u aly MA Name E amp Administrator 6 Folder_o01 LL Experiment_001 Cyt sr Settings 8 ez Global worksheets Global Sheet1 i 53 96 Well U bottom EF 96 Well V bottom 3 Delete unnec
83. iner SIT protector Slide the protector over the SIT and push up on the tube retainer until you can screw it onto the SIT Tighten the tube retainer 6 Attach the HTS sample coupler to the cytometer SIT Slide the sample coupler onto the SIT until you reach a hard stop Make sure the sample coupler tubing is not kinked or twisted Hold the coupler with one hand while you tighten the top nut with the other hand Note that there should be a gap between the tightening nut and the bottom of the SIT protector Figure 3 4 on page 63 If you don t see a gap unscrew the tube retainer push the SIT protector all the way up and retighten the tube retainer 62 BD High Throughput Sampler User s Guide Figure 3 4 Installing the sample coupler on the BD LSR II SIT protector gap tightening nut sample coupler NOTICE Make sure the sample coupler is securely connected to the SIT 7 Turn on the HTS The power switch is on the right side of the HTS unit See Figure 3 5 Figure 3 5 Power switch power switch Chapter 3 Running Samples 63 Creating Experiments An experiment is a group of elements used to acquire and analyze data from the BD High Throughput Sampler The Browser is where you create experiments and access stored data Here are two ways to add experiments to the Browser e Use the New Experiment button f in the Browser toolbar to create a new empty experiment with default experiment elements
84. information available e product name and serial number e any error messages e details of recent system performance For cytometer support from within the US call 877 232 8995 For support from within Canada call 888 259 0187 Customers outside the US and Canada contact your local BD representative or distributor If you need to send your HTS unit back to BD Biosciences for repair see Appendix B on page 157 for instructions Note that depot repair procedures might be different outside of the United States Contact your local BD Biosciences service representative for information for your region About This Guide xi xii BD High Throughput Sampler User s Guide Introduction The BD High Throughput Sampler HTS is a compact high speed sample loading device for use with the BD LSR II BD FACSCanto and BD FACSCanto II flow cytometers BD FACSDiva software controls the sample loader providing automated acquisition of samples from a multiwell plate Automate analysis using the BD FACSDiva batch analysis feature for an efficient high throughput system The following topics are covered in this chapter e HTS Hardware Overview on page 14 e Components on page 16 e Cytometer Connections on page 19 e Sample Processing on page 23 13 HTS Hardware Overview Easy to use and maintain and highly reliable the HTS provides the following basic functionality e Acquires samples from 96 and 384 well plates standard depth
85. lacing HTS Unit into Long Term Storage Unscheduled Maintenance eee eee eee Homing the Sample Probe 0002 eee Priming the HTS 0 0 eee eee ee ee eee Performing a Motion Test 0 000 e ee eeee Verifying the Sample Probe Position Reinitializing the HTS 0 c ce eee eee Inspecting Thumbscrew Fittings 4 Inspecting Hexagonal Fittings Declogging the SIT 0 0 cece cee eee eee Chapter 5 Troubleshooting HTS Troubleshooting 0 00 e cece nee Acquisition Troubleshooting 02 2 ee eee ee BD FACSDiva Troubleshooting 0000 eee Appendix A Consumables and Replacement Parts Cytometer Supplies 0 2 0 c cece ee eee eee Maintenance Log sirena ad aed eee ee Contents vii Appendix B Depot Repair Procedures 157 Depot Repair OvervieW Wesana rE Vie pet eeepc eis sea ages 158 Removing the HTS Unit 2 1 0 0 ccc eects 159 Cleaning the HTS Unit ee peri ieee bch s Seb ei oars YEO he ee 162 Decontaminating the Fluidics Manually 0 0 cece ee eens 163 Decontaminating External Surfaces 00 c cece eee eee eens 167 Unpacking the Replacement Unit 0 0c e ce cece cece tenes 169 Lifting Heavy Objects 0 0 cc ccc ete da 169 Unpacking the HTS Unit 0 0 cee ee eee eens 170 Installing the HTS Unite eer sede ee hae Se Sas BRI Bhan es 170 Packing the Unit fo
86. letely being careful not to overfill Connect the line declogger tool to the waste connector on the back of the unit Slowly pull back the plunger of the declogger tool until all of the DI water is drawn into the barrel Repeat step 1 and step 3 Disconnect the declogger tool and discard the DI water Carefully dry the declogger tool and return it to the HTS accessory kit Remove and discard the secondary pump valve tubing A Appendix B Depot Repair Procedures 165 e Disconnect the top hex screw B from the secondary pump using the 5 16 in wrench in the accessory kit Figure B 5 Figure B 5 Hex screw on secondary pump secondary pump valve tubing A hex screw B secondary pump e Turn the sample injection port wash station C slightly to disengage the magnetic force and then lift it up to expose the thumb screw D on the sample injection tubing E and the thumb screw F on the secondary pump valve tubing A Figure B 6 Figure B 6 Accessing the tubing sample injection port wash station C thumb screw D sample injection tubing E connector G thumb screw F e Unscrew the thumb screw F from the connector G and remove the secondary pump valve tubing A Figure B 6 166 BD High Throughput Sampler User s Guide e Discard the secondary pump valve tubing A as biohazardous waste A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal dise
87. mber of cycles 40 optimizing 26 speed 39 volume 25 39 Index 179 modes 14 23 default HTS settings 40 default settings 37 fluidic 14 high throughput 23 37 plate 19 60 soft standby 23 standard 23 37 tube 19 94 monthly cleaning BD FACSCanto 113 BD LSR II 111 113 maintenance 111 114 motion test 138 N number of mixes 40 0 optimizing cytometer settings 78 80 ordering information howto 151 plates 66 replacement parts 151 153 O rings replacing quick connector 130 SIT protector 130 overflow reservoir 18 P packaging for shipping 175 panel template 85 part replacement air filter 121 hexagonal fittings 143 injection tubing 116 instrument supplies 152 ordering information 151 153 O rings 130 part numbers 152 plates 66 probe 129 pump syringes 125 reagents 153 sample coupler and tubing 123 schedule 114 sheath filter 119 SIT protector 130 thumbscrew fittings 142 perform compensation setup 40 perform panel analysis 40 phone numbers ordering parts 151 technical support xi plate See also plates filter 33 holder inspection and service 110 shown 17 18 79 icons 35 layout view 35 legend 33 mode 19 60 rearranging samples on 44 template 76 toolbar 34 Plate Interface shown 31 180 BD High Throughput Sampler User s Guide plate based acquisition preparing for 59 running samples 55 85 setting up 60 63 vs tube based 22 plates compatibility 66 list of tested 66 ordering information 66
88. n a global worksheet 1 Create an FSC vs SSC plot on the global worksheet The plot parameters should be FSC A and SSC A Create a PE histogram next to the FSC vs SSC dot plot change the plot parameter to PE A Create the following dot plots choosing the appropriate parameters e FITC vs PE e PerCP Cy5 5 vs PE Cy7 e APC vs APC Cy7 Show a population hierarchy The worksheet should look similar to Figure 3 12 on page 75 BD High Throughput Sampler User s Guide Figure 3 12 Plots on global worksheet Global Worksheet Acquisition B SA K6U eS Pee aeeecniBeys N A Atm amp g eR Acquisition Acquisition 100 150 200 250 FSC A 1 000 Acquisition Acquisition 2 5 wo tot 10 PerCP Cy5 5 A 10 Acquisition Tube Acquisition 10 Population Events Parent Total BB All Events O AHE HABE 4 10 APC Cy7 A 10 Chapter 3 Running Samples 75 Mi Tip Alternatively you can view the unstained normal worksheet and choose Edit gt Select All gt Copy Then view the global worksheet right click and choose Paste Draw your gates FSC vs SSC plot and all histograms or you can copy the gate from an unstained normal worksheet During acquisition drag the gate to the population of interest 5 Assign a name to the global worksheet In the Browser select the worksheet then select Rename from the shortcut menu You are now ready to set up
89. n fittings if required See Inspecting Thumbscrew Fittings on page 142 Periodic Maintenance 114 To ensure optimal performance of your HTS system replace the following cytometer components according to the recommended replacement schedule in Table 4 1 Note that the extent of maintenance will vary depending on how much you use your system Use the schedule in Table 4 1 as a guideline A maintenance log is provided in Appendix A to keep track of when each procedure is performed Table 4 1 Recommended replacement schedule Component Part No Replacement Schedule Procedure Sample injection tubing 335526 A every 6 months page 116 335451 F Sheath filter 335710 every 6 months page 119 Air filter 336218 yearly or when needed page 121 Sampler coupler and 335452 yearly or when needed page 123 tubing BD LSR II BD High Throughput Sampler User s Guide Table 4 1 Recommended replacement schedule continued Component Sampler coupler and tubing BD FACSCanto and BD FACSCanto II Syringes Probe Quick connector O ring SIT protector and O ring BD LSR II Long term storage Part No 339340 339047 34389017 343618 335345 Replacement Schedule yearly or when needed yearly or when needed yearly or when needed yearly or when needed yearly or when needed when needed Procedure page 123 page 125 page 129 page 130 page 130 page 132 a See Figure 4 5 on page 116
90. ndling cytometer components Wear suitable protective clothing eyewear and gloves e Primary pump valve to probe There are male thumbscrew fittings at both ends of the tubing e Sample injection tubing There is a male thumbscrew fitting at the sample injection port wash station and a female thumbscrew fitting at the extension tubing to the secondary pump valve e Extension tubing to the secondary pump valve There is a male thumbscrew fitting at the sample injection tubing end 142 BD High Throughput Sampler User s Guide Inspecting Hexagonal Fittings Hexagonal male fittings are located at the following locations Check and tighten these fittings periodically and replace them when needed A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling cytometer components Wear suitable protective clothing eyewear and gloves e Left port of primary pump e Top left and right ports of secondary pump NOTICE Valves contain sealing washers in each port Over tightening fittings can compress the seal washers resulting in a blocked port Insert a fitting or syringe into a port until it contacts the seal washer then turn the fitting or syringe an additional 1 6 to 1 4 turn If leakage is observed tighten a maximum of an additional 1 8 turn Declogging the SIT Perform this procedure to dislodge a clog in the SIT only when instructed t
91. ning sample that was aspirated 46 BD High Throughput Sampler User s Guide e Pause Resume toggle button pauses the sequence after finishing collection of the current well resumes a paused sequence with the next well in the sequence During an uninterrupted sequence BD FACSDiva software calculates the acquisition time of each well based on sample volume This acquisition time is referred to as the stopping time and is calculated as follows sample volume pL flow rate uL sec x 1 sec 1000 msec stop time msec The software will stop acquisition or recording of a well and proceed to the next well when any of the following stopping rules have been met e the specified number of events were collected e the stopping time was reached e the file exceeded memory specifications NOTICE If an HTS error occurs during a run the run will abort and an error message will display Once the sequence run is complete a dialog will be displayed with the status of the run and whether any errors occurred An alert will sound until the dialog is dismissed Running Wells Manually When running samples in manual mode use the Basic Controls in the Acquisition Dashboard You can acquire record and restart acquisition on a single selected well as long as a sequence is not currently running For detailed information on the Basic acquisition controls refer to the BD FACSDiva Software Reference Manual To start manual mode do one of the f
92. nsing is in progress this can take up to 30 minutes 112 BD High Throughput Sampler User s Guide 12 Click OK when monthly cleaning is complete Monthly Clean State G Monthly clean is complete NOTICE If you have been running BD FACSRinse solution as part of a biweekly cleaning procedure you can repeat the monthly cleaning using BD FACSRinse solution followed by DI water 13 Disconnect the tank fill it with sheath solution and reconnect it 14 Reconnect the BD LSR II sheath filter NOTICE If you are performing a sheath fluid exchange install a new filter 15 Check for bubbles and prime if necessary BD FACSCanto and BD FACSCanto II Monthly Cleaning 1 Ensure the sample coupler is properly installed and the HTS is powered on 2 Empty the waste tank if necessary 3 Ensure the BD FACSClean and BD FACS shutdown solution cubitainers are full and connected 4 Choose Cytometer gt Long Clean 5 Click OK when the Long Clean is complete Chapter 4 Maintenance 113 Surface Inspection and Cleaning A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling cytometer surfaces Wear suitable protective clothing and gloves e Use BD FACSClean or a 10 bleach solution followed by DI water to wipe down the HTS enclosure and cover on a monthly basis or when needed e Inspect the pump syringes for leaks and tighte
93. nstalled on the SIT while the aspirator arm rests against the sample coupler Flip the aspirator arm bar to the back as shown in Figure 1 8 on page 22 to ensure it does not come in contact with the HTS probe This position also ensures that the aspirator arm is able to detect an installed sample coupler Chapter 1 Introduction 21 Figure 1 8 Cytometer SIT during plate based acquisition BD LSR II left BD FACSCanto right and BD FACSCanto II bottom SIT protector sample sample coupler coupler aspirator arm aspirator arm bar SIT protector sample aspirator aspirator arm bar e For instructions on switching the cytometer from tube to plate acquisition see Setting Up for Plate Based Acquisition on page 59 e For instructions on switching back from plate to tube acquisition see Returning to Tube Based Acquisition on page 94 22 BD High Throughput Sampler User s Guide NOTICE BD LSR II Soft standby mode is not available when you are acquiring samples in Plate mode Refer to your BD LSR II User s Guide for information about soft standby mode Sample Processing During acquisition the HTS processes samples differently based on the throughput mode Table 1 2 compares acquisition in each mode Table 1 2 Comparison of acquisition sequence in standard and high throughput mode Sequence Standard Mode 1 e raises the probe to the cleaning position e flushes the probe using the primary pump e
94. o do so by BD Biosciences A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling cytometer components Wear suitable protective clothing eyewear and gloves Materials cleaning stylus A To avoid a service call do not insert the cleaning stylus all the way into the SIT allow at least an inch of the stylus to protrude from the end of the SIT while declogging Chapter 4 Maintenance 143 1 Remove the sample coupler 2 About an inch from one end of the cleaning stylus bend it at a 90 angle Bending the end of the cleaning stylus will prevent you from inserting it too far into the SIT 3 Hold the bent end of the cleaning stylus and thread about three quarters of the stylus up into the SIT see Figure 4 19 Figure 4 19 Declogging the SIT SIT cleaning stylus 4 Gently pull the cleaning stylus most of the way out of the SIT and then push it back in repeat several times 5 Remove the cleaning stylus from the SIT rinse the stylus with BD FACSClean followed by DI water and dry the stylus 6 Replace the sample coupler 7 Choose HTS gt Prime 144 BD High Throughput Sampler User s Guide 5 Troubleshooting The tips in this section are provided to help you troubleshoot issues that might arise when using the BD High Throughput Sampler For additional troubleshooting assistance refer to the appropriate cytomet
95. o the reservoir see Figure 1 4 on page 18 8 Connect the sheath line with filter and waste lines to their respective ports on the back of the unit or back of the cytometer for BD FACSCanto II see Figure B 10 NOTICE The white connector on the BD FACSCanto II is not used for the HTS 172 BD High Throughput Sampler User s Guide Figure B 10 Connecting BD FACSCanto II interface cable to cytometer sheath line blue waste line orange interface communication cable BD FACSCanto Install the rear catch tray Route the sample coupler underneath the catch tray tongue and sample coupler slot Push the tray in making sure the coupler line is not crimped or kinked The clamp on the tray is used for the sample coupler when it is not in use Figure B 11 Catch tray installed at the back of the BD FACSCanto clamp for sample coupler Appendix B Depot Repair Procedures 173 10 11 12 A Slide the HTS unit all the way back toward the cytometer being careful not to crimp the fluid tubing or communication cable If necessary install the probe refer to Replacing the Probe on page 129 Connect the sample coupler to the SIT BD LSR II With the SIT protector in place slide the sample coupler onto the SIT until you reach a hard stop Make sure the sample coupler tubing is not kinked or twisted Hold the coupler with one hand while you tighten the top nut with the other hand sample coupler BD LSR
96. ollowing e Click on a well and then click Acquire Data under Basic Controls to start acquisition Chapter 2 BD FACSDiva Software Overview 47 48 e Click on a well and then click Record Data under Basic Controls to start recording data NOTICE While running plate based acquisition the Start Acquisition on pointer change preference in the User Preferences window is ignored and the software proceeds as if the preference were set to Off NOTICE During manual acquisition high throughput mode is not applicable Stopping time in manual acquisition mode is calculated the same as in automatic sequence mode The software will stop acquisition or recording of a well when any of the following stopping rules have been met e the specified number of events were collected e the stopping time was reached e the file exceeded memory specifications e acquisition was stopped by clicking the Stop Acquiring button under Basic Controls Once acquisition of the well is complete any remaining sample is flushed from the system The controls in the Acquisition Dashboard are briefly disabled while the loader is reset Analysis View Components The Analysis view provides an interface for e displaying keyword results in the plate layout e reviewing and analyzing data collected from a plate e starting a batch analysis For information on performing these functions see Analysis View Functions on page 51 BD High Throughput Sampler User s Guide
97. ometer Settings Select the well you wish to copy to then right click it and choose Paste Cytometer Settings Chapter 2 BD FACSDiva Software Overview 45 Acquisition Mode There are two ways to run plate based acquisition on the HTS automatically in sequence or manually one well at a time See Running Samples Automatically in Sequence in the following section and Running Wells Manually on page 47 for a description of each mode Running Samples Automatically in Sequence When running samples in sequence use the HTS Controls in the Acquisition Dashboard Data collection proceeds automatically according to sequence number Select noncontiguous samples by holding down the Ctrl key while clicking individual specimens or select a range of samples using the mouse Wells are run in the following order e setup controls e compensation controls e specimen wells NOTICE When a sequence is in progress the ability to select a well in the plate layout is disabled During a sequence the acquisition buttons have the following function e Run Plate Stop Plate toggle button starts recording data for all wells in the plate terminates the sequence after recording data for the current well The system flushes any remaining sample that was aspirated e Run Well s Stop Well s toggle button starts recording for all wells selected in the sequence terminates the sequence after recording data for the current well The system flushes any remai
98. ometers For an in depth description of software components not described in this chapter refer to the BD FACSDiva Software Reference Manual The following topics are covered in this chapter e Workspace Components on page 28 e Plate Window on page 32 27 Workspace Components 28 When you start BD FACSDiva software the workspace appears Figure 2 1 Windows containing the main application components are displayed within the workspace Display additional windows by clicking buttons in the Workspace toolbar Figure 2 1 BD FACSDiva workspace displaying an open experiment and the Plate window BD FACSDiva Software Administrator File Edit View Experiment Populations Worksheet Cytometer Carousel HTS Help agde x E Globat Worksheet Global Sheet1 Bo Zik W lA EAA n E S e E aE e X 3 Cytometer FACSCantoll 1 rE NNE Sl i a ot g h Name Dae Jobal Sheet B Administrator S Folder_oo1 S 002 D6 ll Experiment_001 2 1 07 1 30 31 PM E Inspector Dot Plot EA eee Oe pe Bl Blank Experiment with Sam 2 1 07 1 25 53 PM a i 5 9 Cytometer Settings FE Te Label acaistion Dot Pot Zg ma m oes amp ai renee Tube U bottom Specimen_002 06 lt 2 s re oa Neu boon res nae f 2 32 S cF_28_tan 2007 2 1407 2 25 27 PM spaa eee fpa h Sn 9 cytometer Settings Y
99. op nut with the other hand Allow the aspirator arm to rest against the sample coupler NOTICE Flip the aspirator arm bar backwards to ensure it does not come in contact with the HTS probe This position also ensures that the aspirator arm is able to detect an installed sample coupler Figure 3 2 Installing the sample coupler on the BD FACSCanto or BD FACSCanto II sample coupler aspirator arm moved towards back 5 Prime the HTS twice by choosing HTS gt Prime NOTICE Make sure the sample coupler is securely connected to the SIT Setting Up for Plate Based Acquisition on the BD LSR II When switching from tube to plate mode you need to place the acquisition control switch in plate mode and replace the DCM sleeve with the sample coupler as described in this section BD High Throughput Sampler User s Guide 5 If necessary install the HTS unit For instructions see Installing the HTS Unit on page 170 a a ooe Switch the acquisition control switch to plate mode 88 ee Remove the tube of DI water from the SIT Remove the DCM sleeve Unscrew the tube retainer that holds the DCM sleeve onto the SIT and carefully remove the sleeve tube retainer DCM sleeve Install the SIT protector The SIT protector is a modified sleeve that prevents the sample injection tube from bending during installation of the sample coupler Chapter 3 Running Samples 61 Figure 3 3 Installing the SIT protector tube reta
100. orienting 79 printing records 83 88 sizes supported 14 working with 35 position test 140 probe assembly 17 18 homing 136 inspection and service 110 preventing damage 78 replacing 129 verifying position 140 wash volume 39 pumps inspection and service 110 replacing syringes 125 secondary shown 17 18 Q quitting software 101 R rate sample flow 39 records acquisition status 36 83 printing 86 renaming specimen ina plate 42 repair procedures about 158 cleaning the HTS unit 162 168 decontaminating exterior 167 decontaminating fluidics 163 installing replacement unit 170 packing unit for shipping 175 removing the HTS unit 159 rinsing fluidics 165 unpacking replacement unit 169 replacement unit unpacking 169 returns packaging 175 run sequence automatic 46 manual 47 S safety biological 59 160 drip containment module DCM 19 interlock feature 82 lifting heavy objects 169 maintenance procedures 103 144 tube based acquisition 95 unpacking replacement unit 169 sample coupler 21 23 BD FACS Canto II 18 BD LSRII 17 inspection and service 109 sample volume 39 samples aspirated volume 39 flow rate 39 minimum volume 39 processing overview 23 running 55 85 sample injection tube SIT 21 setting volume 25 40 servicing cytometer 109 Index 181 settings carryover 26 HTS 38 mixing 26 optimizing for acquisition 78 throughput 26 Setup view components 33 functionality 43 sheath filter replacing
101. ormation on acquisition stopping criteria see Running Samples Automatically in Sequence on page 46 and Running Wells Manually on page 47 44 BD High Throughput Sampler User s Guide Applying an Analysis Template You can apply an Analysis template to selected samples or wells First select the well then e choose Edit gt Apply Analysis Template in the menu bar or e right click to select Apply gt Analysis Template in the shortcut menu For instructions on how to save an Analysis template see Exporting an Experiment as a Template on page 84 Applying HTS Settings Apply specific HTS settings to a well or group of wells by using the Copy Paste Loader Settings commands in the shortcut menu Copying and Pasting Cytometer Settings You can copy and paste cytometer settings from one specimen or well to another as long as the specimen or well that you are copying from has cytometer settings You can copy and paste cytometer settings to and from unrecorded wells only To copy settings from one specimen to another click to select the specimen in the specimen list see Figure 2 2 on page 32 then right click the selected specimen on the plate and choose Copy Cytometer Settings Select the specimen you wish to copy to in the specimen list then right click the selected specimen on the plate and choose Paste Cytometer Settings To copy settings from one well to another click to select the well then right click it and choose Copy Cyt
102. ort gt Experiment Template Vv Tip Make sure you choose the Experiment Template command and not the Experiments command The Export Experiment Template Wizard appears The bold text at the top of the dialog tells you what to do at each screen Select Template Type and enter a Template name Type Fenerai gt Name Experimento T Lock Template ae ee 3 Enter Practice for the template Type and 6 Color Analysis for the template Name The template Type is used to group similar templates so they are easy to find At a minimum you need to enter only the Type and Name when you are creating a template the remaining screens are optional Vv Tip Select the Lock Template checkbox when you want to prevent other users from overwriting your template with the same name 4 Click Next Next and then Finish to view the remaining screens Vv Tip To export a Panel template select a specimen in the Plate window and follow the steps above Chapter 3 Running Samples 85 Analyzing Data This section shows you how to use Analysis view and the batch analysis feature in BD FACSDiva software to analyze data from plate based acquisition Displaying Keywords 1 Click the Analysis tab to display the Analysis view The Analysis view shows the acquisition status of each well E Plate 96 Well U bottom _ Setup Analysis Legend wily Specimen type BI First well in group Keywords Specimen number aie
103. otector straight down A To avoid bending the SIT do not slide the SIT protector at an angle 5 Reinstall the standard DCM sleeve Figure 3 19 on page 95 Slide the sleeve straight up over the SIT and screw the tube retainer on to secure it 94 BD High Throughput Sampler User s Guide Figure 3 19 Installing the DCM sleeve tube retainer DCM sleeve Install a tube of DI water on the SIT and place the tube support arm under the tube Switch the acquisition control switch to tube mode W If necessary detach the HTS unit from the cytometer see Removing the HTS Unit on page 159 and Figure 3 20 on page 96 and remove the base plate by loosening the thumb screws at the bottom of the cytometer support bracket see step 13 on page 135 If you will not be using the HTS for a long period of time follow the instructions under Placing HTS Unit into Long Term Storage on page 132 To prevent personal injury or damage to the sampler disconnect the BD High Throughput Sampler from the cytometer and remove the unit before running tube based acquisition Chapter 3 Running Samples 95 Figure 3 20 HTS unit detached from cytometer cytometer support bracket thumb screw thumb screw base plate 9 If applicable perform the monthly cleaning as outlined in Monthly Cleaning on page 111 install a new sheath filter and fill the sheath tank with BD FACSFlow solution Exchanging the Sheath Fluid on a BD LSR II Follow the in
104. p and then moves to well A1 The following message appears Position Test 4 Postion Am Click OK to continue 5 Remove or open the safety cover and verify that the probe is in the center of the nearest well position A1 6 Replace or close the safety cover and click OK The probe moves to well H12 and the following message appears Position Test 4 Postion H12 Click OK to continue 7 Remove or open the safety cover and verify that the probe is in the center of the farthest well position H12 8 Replace or close the safety cover and click OK The probe returns to the injection port and the following message appears Position Test G Position Test Completed 9 Click OK to dismiss the message Chapter 4 Maintenance 141 Reinitializing the HTS If you switch off the HTS and then switch it back on again while the software is running you must reinitialize the HTS 1 Choose HTS gt Reinitialize The following message appears when reinitialization is complete Relnitialize G HTS Re initialization Complete 2 Click OK to dismiss the message Inspecting Thumbscrew Fittings Thumbscrew fittings are installed in the following tubing locations Check and tighten these thumbscrews periodically and replace them when needed A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when ha
105. pler rrer ririri koet Eaa 8 eke eee eee Lae EEEE ERGS Sample Processing pra s sceccebon Bai Ge R Gg ee eR LES ode ea RS ae Chapter 2 BD FACSDiva Software Overview Workspace Components Plate Window 0 eee ee eee Setup View Components Setup View Functions Analysis View Components Analysis View Functions Chapter 3 Running Samples Starting Up get sce ss ae aa aes Setting Up for Plate Based Acquisition Xi 13 14 16 19 19 21 23 27 28 32 33 43 48 51 55 56 59 Creating Experiments tesi rria En eee eee eee tence nnes Creating a Folder and an Experiment 0 000 eee eeuee Setting Up the Plates ccc cuka ected ae te kanonan eres Setting Up the Worksheet 00 ce cece eee eee eens Exporting a Plate asa Template 0 0 cece eee eee Assigning Keywords 00 cece cee eee nent eens Preparing for Acquisition 0 e cece cece cece ence eens Optimizing Cytometer and HTS Settings 0 000 eee Performing Compensation 000 cece cece eee eens Acquiring Data epua ato anaoa Geibd E8era a a Perea diere Sas Pausing the BD High Throughput Sampler 0000 Stopping the BD High Throughput Sampler 065 Exporting an Experiment as a Template 2000 Analyzing Datars es smien clea ae ster ewes dae Ghee Ne at Displaying Keywords 0c cece cece cette ences
106. plete Click OK to dismiss the completion message BD High Throughput Sampler User s Guide 5 Open the safety cover and remove the plate NOTICE Access the plate only after sample processing is complete and the probe is no longer moving 6 Click the Print button in the Plate toolbar to print a record of the Setup view and legend A Keep a record of acquisition status by printing a copy of the Setup view immediately after acquisition is complete Refer to the printout if you reanalyze or export the data If you export a data file or an experiment that contains incomplete data the software cannot recognize that the file is incomplete 7 Select the appropriate settings and printer and then click OK Use the printout to e view acquisition run order according to well numbering e determine well status post acquisition See Plate Layout on page 35 8 At the end of each day or shift clean and service the cytometer as described in Daily Maintenance on page 104 Pausing the BD High Throughput Sampler To pause during a run click Pause pase in the Acquisition Dashboard The HTS unit finishes processing the current well or wells in high throughput mode and then remains in a suspended state until you choose to continue e To continue the run click Resume e To stop the run click Stop Well s _ stopwews or Stop Plate _ stopriete This sequence allows you to stop the run early to minimize additional sample loss
107. power to the cytometer Quit the software see Quitting the Software on page 101 Fluidics Shutdown BD FACSCanto and BD FACSCanto II Perform a fluidics shutdown before quitting the software 100 BD High Throughput Sampler User s Guide 1 Ensure that the sample coupler is attached NOTICE Always run the fluidics shutdown with the sample coupler attached to rinse the syringes and fill them with BD FACS shutdown solution 2 Choose Cytometer gt Fluidics Shutdown The following dialog appears Confirm Fluidics Shutdown can take up to 5 min Click OK to initiate Click Cancel to abort 3 Click OK A dialog appears when fluidics shutdown is complete Ej Shutdown Status wD Fluidics shutdown is complete It is now safe to turn off your system 4 Click OK Quitting the Software If you have a BD FACSCanto and BD FACSCanto II you must perform a fluidics shutdown before quitting the software see Fluidics Shutdown on page 100 1 Choose File gt Quit All Browser and worksheet elements are automatically saved when you quit the application Chapter 3 Running Samples 101 102 BD High Throughput Sampler User s Guide 4 Maintenance This chapter provides maintenance procedures to keep your HTS in good working order The following maintenance procedures are covered in this chapter e Daily Maintenance on page 104 e Monthly Maintenance on page 111 e Periodic Maintenance on p
108. quisition order Table 2 1 Assessing well status during setup or acquisition Well Status selected for acquisition if selected for acquisition and contains previously recorded data il Chapter 2 BD FACSDiva Software Overview 35 Table 2 1 Assessing well status during setup or acquisition continued Well Status acquiring recorded selected for acquisition and contains previously recorded data from aborted run selected for acquisition aspirated and aborted a The error message is cleared from the Status tab of the Cytometer Settings window when acquisition begins Table 2 2 Assessing well status once acquisition is complete Well Status well contains data acquisition successful well contains no data well contains data recording aborted well contains no data acquisition aborted A To keep a record of acquisition status print a copy of the Setup view immediately after acquisition is finished and refer to the printout if you reanalyze or export the data If you export a data file or experiment that contains incomplete data the software cannot recognize that the file is incomplete 36 BD High Throughput Sampler User s Guide Compensation Controls You can create label specific compensation controls Refer to the BD FACSDiva Software Reference Manual for information Throughput Mode The default mode at startup is high throughput Select throughput mode
109. r List of specimens on the plate 4 Specimen_001 2 Setup Controls_001 EJ Compensation Controls Cut Copy and Paste Features You can copy and paste well setup and elements to a blank well The pasted wells inherit the loader and cytometer settings from their respective sources This does not apply to the compensation controls specimen because it is unique to the experiment To copy and paste wells 1 In the Plate view select the wells to be copied 2 Inthe shortcut menu choose Copy gt Well s 3 Click to set the paste direction to horizontal or vertical This automatically adds a new well to the existing specimen in the direction selected or To paste to a different area of the plate and make a new specimen select an empty well From the shortcut menu choose Paste gt Well s 42 BD High Throughput Sampler User s Guide To copy and paste a specimen 1 Click the specimen in the List of specimens on the plate Right click a well in that specimen then select Copy 2 Select an empty well then right click it and select Paste The specimen will be pasted if there is room on the plate Setup View Functions Use the Setup view to set up new plate based experiments Once you have added setup controls samples and cytometer settings to the plate layout you can assign keywords set recording rules apply a selected assay Analysis template and copy and paste HTS loader settings to selected wells o
110. r Shipping 0 ccc cece eee eee eens 175 Placing the HTS into Its Shipping Container 0 00000 eee 175 Index 177 viii BD High Throughput Sampler User s Guide About This Guide This guide describes how to set up and operate the BD High Throughput Sampler HTS option with the BD LSR II BD FACSCanto and BD FACSCanto II flow cytometers You should know how to operate your flow cytometer before using the HTS option For important safety information refer to the safety booklet provided with the HTS option Cytometer function is controlled by BD FACSDiva software In this guide you will find a description of BD FACSDiva software features specific to the HTS option BD LSR II BD FACSCanto and BD FACSCanto II flow cytometers modified with the HTS can acquire samples from plates or tubes Even when acquiring samples using the HTS consult the appropriate cytometer user s guide for information about flow cytometer operation daily shutdown maintenance and troubleshooting The BD High Throughput Sampler User s Guide assumes you have a working knowledge of basic Microsoft Windows operation If you are not familiar with the Windows operating system refer to the documentation provided with your computer Conventions The following tables list conventions used throughout this guide Table 1 lists the symbols that are used in this guide or on safety labels to alert you to a potential hazard
111. r to the entire plate Use the Setup view to perform the following functions e Define loader settings e Optimize cytometer settings while running setup controls e Monitor well status during acquisition e Acquire and record wells manually or automatically e Analyze plate data Chapter 2 BD FACSDiva Software Overview 43 Rearranging Samples on the Plate Rearrange samples and wells in the plate layout by using the Cut Copy and Paste functions Note that specimens and wells cannot be overwritten e You can move specimens to different locations in the plate layout as long as there is room to accommodate the entire specimen e You can move wells within a specimen to change the acquisition order or move wells from one specimen to another Wells are pasted either below or to the right of the selected well depending on the orientation of the Add Well button Assigning Keywords You can create and assign keywords at the experiment or well level For information on creating experiment keywords refer to the BD FACSDiva Software Reference Manual For instructions on creating and assigning keywords at the well level see Assigning Keywords on page 77 Setting Recording Rules You can alter the acquisition stopping criteria for a selected well by e changing the Sample Flow Rate and Sample Volume in the HTS tab of the Well Inspector e changing the Events to Record in the Experiment Layout or Acq tab of the Well Inspector For inf
112. rce of your legs Do not twist your back when lifting pivot on your heels Unpacking the HTS Unit 1 With the arrows on the box pointing up open the shipping container Remember to save all paperwork and shipping materials to repack the unit to be returned Remove the top piece of foam and set it aside Using proper lifting techniques carefully lift the unit out of the container and place it on a table near the cytometer The unit is encased in plastic to protect it during shipment The plastic can make the cytometer slippery and difficult to grasp To prevent personal injury or damage to the cytometer use caution when lifting the cytometer out of the container Remove the HTS unit from the plastic bag Remove the small I shaped piece of foam that secures the HTS probe assembly and plate holder and set it aside Installing the HTS Unit Follow this procedure to install your replacement HTS unit A Any cytometer surface that comes in contact with biological specimens can 1 transmit potentially fatal disease Use universal precautions when handling cytometer hardware Wear suitable protective clothing and gloves To avoid damaging the HTS probe 170 BD High Throughput Sampler User s Guide 2 e lower it into the injection port wash station e BD FACSCanto II remove the probe and the overflow reservoir See Replacing the Probe on page 129 BD FACSCanto II Open the doors to the enclosure and place t
113. rrors or omissions nor for any damages resulting from the application or use of this information BD Biosciences welcomes customer input on corrections and suggestions for improvement BD FACSDiva software 2007 Becton Dickinson and Company This software is the property of Becton Dickinson and Company Each sale of a stored unit of this software grants the purchaser a nontransferable nonexclusive personal license This software may not be duplicated reproduced or copied in any form or by any means whatsoever except as otherwise permitted by law This product includes software developed by the Apache Software Foundation apache org BD BD logo and all other trademarks are property of Becton Dickinson and Company Adobe and Acrobat are registered trademarks of Adobe Systems Incorporated Diskeeper is a registered trademark of Executive Software International Microsoft and Windows are registered trademarks of Microsoft Corporation Sybase Adaptive Server Anywhere and SQL Anywhere are trademarks of Sybase Inc or its subsidiaries Cy is a trademark of Amersham Biosciences Corp Cy dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use Any other use requires a commercial sublicense from Amersham Biosciences Corp 800 Centennial Avenue Piscataway NJ 08855 1327 USA
114. s Legend Keywords we Specimen type First well in group kz Specimen number Name Suffix 2 2 Lymphocytes E Granulocytes List of specimens on the plate EI Setup Controls_001 E Compensation Controls 13 Specimen_001 4 Specimen_002 6 Click the Print button 8 in the Plate toolbar to print the Concentration Keyword view and legend 7 Choose the appropriate print options and click OK Save the printout for your records 8 Click the keyword Lymphocytes in the Keyword list The Keyword view displays the values for the keyword Lymphocytes See Figure 3 17 on page 89 88 BD High Throughput Sampler User s Guide Figure 3 17 Keyword Analysis view showing Lymphocytes values Plate 96 Well U bottom Setup Analysis Legend Keywords ea Specimen type pE First well in group bz Specimen number Name Suffix BB Concentration o Lymphocytes DB Granulocytes List of specimens on the plate H Setup Controls_001 AM Compensation Controls 3 Specimen_001 4 Specimen_002 9 Print the Lymphocytes Keyword view and legend 10 Click the keyword Granulocytes in the Keyword list The wells that were designated with the keyword Granulocytes are colored yellow green but do not list a value since none was assigned Chapter 3 Running Samples 89
115. s The HTS goes through a purge cycle If the stopping time is met or the specified number of events are collected acquisition of the current well will stop the HTS will be purged and the software will automatically proceed to the next well NOTICE No data file is saved for setup control wells 80 BD High Throughput Sampler User s Guide Performing Compensation Once you have optimized cytometer settings you are ready to run your compensation controls and calculate compensation NOTICE You can run compensation controls only once during an experiment If you need to re run compensation controls you must create a new experiment 1 2 n OD UOU OW Select wells B1 through B7 in the plate layout Click Run Well s Runwes in the Acquisition Dashboard The compensation wells will be run in the order they were created NOTICE You must select a well s in the Setup view as well as a global worksheet to enable the Run Wells button After the last compensation control has been recorded the Sequence Done dialog appears Sequence Done w The Run Completed Click OK Toggle to the normal worksheet for the compensation control wells Select the FITC Stained Control worksheet in the Worksheet window Verify that the P1 gate encompasses the singlet bead population Verify that the Autointerval gate encompasses the positive peak in each of the plots on all worksheets Choose Cytometer gt Cytometer Setup gt
116. se a keyword the plate layout shows keyword values and corresponding background colors Wells are colored according to keyword assignments For example Figure 2 6 displays values for the keyword Concentration In this example four different concentrations were assigned to wells on the plate The Keyword Analysis view shows the concentration value assigned to each well Figure 2 6 Keyword Analysis view ell U bottom x z Keywords oe Specimen type pE First well in group bz Specimen number Aj a Concentration Lymphocytes 1 2 3 4 5 6 7 8 9 10 11 12 Granulocytes m List of specimens on the plate Nal H Setup Controls_001 2 Compensation Controls F YE T3 Specimen_001 4 Specimen_002 BD High Throughput Sampler User s Guide Analyzing Plate Data Analyze plate data the same way you would analyze tube data For detailed information refer to the BD FACSDiva Software Reference Manual Performing Batch Analysis Use the batch analysis feature to automatically advance a selected set of data for multiple wells through an analysis template on a global worksheet You can also assign a preferred global worksheet to a specimen or individual wells using the Inspector You can set up batch analysis to pause between each data file and to print a copy of the analysis before proceeding with the next well NOTICE To p
117. sh station Insert cleaning stylus into drain on side of wash station e Replace wash station assembly Sheath float switch time out error during sheath exchange Air in the fluid lines e Choose Cytometer gt Cleaning Modes gt Prime after Tank Refill e Bleed air out of sheath filter 146 BD High Throughput Sampler User s Guide Acquisition Troubleshooting Observation Unexpectedly low event rate or no events in plots Possible Causes Sheath not pressurized Recommended Solutions BD LSR II Ensure that pressure relief valve on cytometer sheath tank is in the closed position Sample injection tubing kinked or damaged Replace the tubing See Replacing the Sample Injection Tubing on page 116 Sampler coupler tubing Replace the tubing See Replacing the kinked or damaged Sample Coupler and Tubing on page 123 Clogged probe Run the daily cleaning procedure to clean the probe See Daily Cleaning on page 104 If the probe remains clogged replace it See Replacing the Probe on page 129 Clogged sample injection tubing SIT Check for clogs in the SIT using the cleaning stylus See Declogging the SIT on page 143 for procedure See Table A 2 on page 153 for cleaning stylus ordering information If the SIT remains clogged replace it See Replacing the Sample Injection Tubing on page 116 Incorrect cytometer settings e Ensure cytometer settings are correct e
118. structions under BD LSR II Monthly Cleaning on page 111 In step 14 replace the sheath filter with a new filter instead of reconnecting the old filter NOTICE If you exchanged BD FACSFlow with BD FACS sheath solution with surfactant install the HTS sample coupler then prime the system twice by choosing HTS gt Prime This will exchange the fluid in the syringes with BD FACS sheath solution with surfactant 96 BD High Throughput Sampler User s Guide Returning to Tube Based Acquisition on the BD FACSCanto and BD FACSCanto II Before running BD FACSCanto clinical software you must exchange BD FACS sheath solution with surfactant with BD FACSFlow Version 2 1 of the clinical software will not connect in acquisition mode if the sheath type is set to BD FACS sheath solution with surfactant 1 Perform a sheath exchange to replace BD FACS sheath solution with surfactant with BD FACSFlow See Exchanging the Sheath Fluid on a BD FACSCanto and BD FACSCanto II on page 98 2 Detach the sample coupler from the cytometer SIT by unscrewing the top thumbscrew Once the coupler feels free gently pull it straight down from the SIT A To avoid bending the SIT pull straight down on the coupler Do not pull the coupler at an angle NOTICE For the BD FACSCanto place the sample coupler in the clamp on the catch tray when the coupler is not in use see Figure B 11 on page 173 Chapter 3 Running Samples 97 Exchanging the Sheath Fluid on a BD F
119. t and the bottom of the SIT protector If you don t see a gap unscrew the tube retainer push the SIT protector all the way up and retighten the tube retainer Placing HTS Unit into Long Term Storage To prevent salt deposits from forming in the fluidics system perform the following procedure if you will not be using your HTS unit for a week or more See Cleaning the HTS Unit on page 162 A All cytometer surfaces that come in contact with biological specimens can 132 transmit potentially fatal disease Use universal precautions when handling cytometer components Wear suitable protective clothing eyewear and gloves Placing the HTS for BD LSR II in Long Term Storage 1 Remove the HTS safety cover 2 Locate and loosen the positioning screw that secures the HTS to the cytometer support bracket Figure 4 17 on page 133 BD High Throughput Sampler User s Guide Figure 4 17 Loosening the positioning screw support bracket positioning screw 3 Grasp the sides of the HTS unit and slide it forward until it meets the stop in the cytometer support bracket 4 Lift the HTS unit off of the cytometer support bracket by doing the following A To prevent personal injury or damage to the HTS unit do not slide the HTS unit out so far that it becomes unbalanced or the strain of the tubing bends the SIT e Slightly lift the HTS unit to clear the positioning screw e Being careful not to strain the sheath tubing slide the unit
120. t to begin batch analysis A message informs you when batch analysis is complete NOTICE A plate GUID globally unique identifier appears in the statistics view This number is unique for each plate Maintaining Data Maintain the database by keeping the size below recommended limits backing up data on a regular basis and deleting experiments and plates that you no longer need Follow the precautions and instructions for database management given in the BD FACSDiva Software Reference Manual Chapter 3 Running Samples 93 Returning to Tube Based Acquisition Returning to Tube Based Acquisition on the BD LSR II To return to tube mode you need to remove the sample coupler and the SIT protector reinstall the DCM sleeve place the acquisition control switch in tube mode and remove the HTS as described in this section A Any cytometer surface that comes in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling cytometer hardware Wear suitable protective clothing and gloves 1 Place the cytometer in Standby 2 Switch off the HTS power 3 Detach the sample coupler from the cytometer SIT by unscrewing the top thumbscrew Once the coupler feels free gently pull it straight down from the SIT A To avoid bending the SIT pull straight down on the coupler Do not pull the coupler at an angle 4 Remove the SIT protector Unscrew the tube retainer and slide the SIT pr
121. te that samples are mixed less effectively in flat bottom wells than U or V bottom wells When using flat bottom wells you might want to mix the sample well before pipetting it into the plate and acquire the sample before it has a chance to settle Carryover You can vary the amount of wash volume to minimize carryover In general a greater wash volume results in less carryover but the greater the volume the slower the system throughput The minimum volume is determined by the sample and mixing volume To start with try a 400 pL wash for 10 pL of sample volume As the sample and mixing volumes decrease the wash volume can also be reduced Throughput The speed at which the HTS completes a plate depends on the acquisition time sample volume sample rate acquisition mode high throughput or standard mixing volume and speed number of mixes and wash volume To achieve optimal throughput BD recommends that you concentrate the sample reduce the sample volume 10 pL or less increase the sample rate 1 pL sec or greater minimize the number of mixes 2 or less and decrease the wash volume 400 pL or less NOTICE The BD FACSCanto II maximum event rate is 10 000 events second BD High Throughput Sampler User s Guide 2 BD FACSDiva Software Overview This chapter describes the BD FACSDiva software features necessary to operate the BD High Throughput Sampler with the BD LSR II BD FACSCanto and BD FACSCanto II flow cyt
122. the cytometer use caution when placing the cytometer in the shipping container 8 Using proper lifting techniques place the bagged unit in the shipping container making sure the arrows on the box are pointing up For proper lifting techniques see Lifting Heavy Objects on page 169 Place the cytometer on top of the bottom piece of foam 9 Place the top piece of foam over the cytometer 10 Lay the Cytometer Return form on top of the foam and close and seal the flaps of the shipping container 176 BD High Throughput Sampler User s Guide Index A acquisition automatic 46 Dashboard 30 data 82 83 defaults 37 40 high throughput mode 23 37 manual 47 modes 23 optimizing settings for 78 plate based See plate based acquisition standard mode 23 37 status 35 status record 83 switching modes 94 time equation 39 troubleshooting 147 tube based 22 94 acquisition status record 36 air filter cleaning 121 Analysis view components 48 functionality 51 analysis automating 13 90 application window components 28 apply analysis template to wells 40 apply application settings 40 apply cytometer settings to wells 40 assistance technical xi available well volume 25 backdripping 19 base plate inspection and service 110 removing 135 batch analysis 90 92 BD High Throughput Sampler See HTS Browser shown 29 C carryover minimizing 26 cleaning air filter 121 daily 104 108 fluidics 163 HTS unit 162 168 Long Clean
123. thumbscrew inspecting 142 tightening 124 133 143 160 fluidic modes fluidics shutdown 101 fluidics startup 58 sheath exchange 99 fluidics cleaning 163 manually decontaminating 163 modes 14 rinsing 165 shutdown BD FACSCanto 100 startup BD FACSCanto 58 tubing shown 17 18 fluorophore labels 37 178 BD High Throughput Sampler User s Guide G GUID 65 93 H hardware basic functionality 14 components 16 installation 15 part replacement 114 plate sizes 14 hazard symbols x hexagonal fittings inspecting 143 High Throughput Sampler See HTS high throughput mode 23 37 HTS decontaminating for repair 162 hardware See hardware installing replacement unit 170 introduction 13 long term storage 132 pausing 83 priming 138 reinitializing 142 settings 39 stopping 84 unpacking replacement unit 169 import well settings 40 injection port interface 17 18 tubing replacing 116 inspection cytometer 109 fittings hexagonal 143 thumbscrew 142 surface 114 Inspector window 31 instrument settings copying and pasting 45 interface panel cytometer 19 21 K Keyword Analysis view feature 52 86 keywords assigning 44 77 viewing assignments 52 86 L labels 72 labels for fluorophores 37 loader settings choosing 25 long term storage of unit 132 M maintenance daily 104 110 log 154 monthly 111 114 part replacement schedule 114 procedures 103 144 unscheduled 136 144 menus software 29 mixing nu
124. towards you until you can access the filter This allows you better access to the connections on the back of the unit 5 Detach the sheath line from Sheath B port on the back of the HTS unit Figure 4 18 on page 134 Press the metal button to release the connector Leave the line attached to the cytometer interface panel Chapter 4 Maintenance 133 Figure 4 18 HTS sheath and waste lines BD HTS sheath line sheath connector 6 Connect the purging assembly line to the Sheath B port The purging assembly line is located in the spares kit 7 Put the end of the purging assembly line into a 500 mL beaker containing DI water Sheath B connector in port purging assembly line beaker of DI water 8 Put the safety cover on the HTS 134 BD High Throughput Sampler User s Guide 10 11 12 13 Choose HTS gt Prime repeat nine times Priming will replace the sheath fluid with DI water Remove the end of the purging assembly line from the DI water and lay it on the benchtop Remove the purging assembly line and reconnect the HTS tubing to the Sheath B port Detach the HTS from the cytometer by following the steps in Removing the HTS Unit on page 159 Remove the HTS base plate e Unscrew the two thumb screws on each side of the base plate cytometer support bracket thumb screw thumb screw base plate e Pull the base plate toward you until it disengages from the cytometer support brack
125. treme positions for a selected plate type e The probe returns to the injection port and the following message appears Motion Test HTS Motion Test Complete Chapter 4 Maintenance 139 5 Click OK to dismiss the message If the motion test fails contact your local BD Biosciences service representative Verifying the Sample Probe Position This test will position the probe at the center of the nearest and farthest wells on a plate so you can visually verify the mechanical positioning of the probe To run this test you must select a plate type in BD FACSDiva software and install the corresponding plate on the plate holder The test can be run with a 96 or 384 well plate 1 Choose HTS gt Motion and Position Test The Motion and Position Test dialog appears 2 Choose the plate type from the drop down menu Motion And Position Test Motion Test 96 Vell Flat Bottom 96 Vell U Bottom 96 Vell V Bottom 384 Well Flat Bottom Y offset 40 H IREF offset 2 100 4 oK After you select the plate type the Position Test button _ Postion test becomes enabled 3 Install the corresponding plate onto the plate holder replace the safety cover and click Position Test Postion test 140 BD High Throughput Sampler User s Guide 4 Verify that the sample probe properly performs the following sequence e The probe travels to the home position e The probe moves u
126. turns on the waste pump e raises the probe e refills the primary pump e turns off the waste pump e draws a separator bubble into the tip of the probe moves the probe and the plate holder so the probe is positioned above the required well lowers the probe and mixes the sample aspirates the required sample volume plus 20 uL of dead volume required by the system along with a separator bubble High Throughput Mode e raises the probe to the cleaning position e flushes the probe using the primary pump e turns on the waste pump e raises the probe e refills the primary pump e draws a separator bubble into the tip of the probe moves the probe and the plate holder so the probe is positioned above the required well lowers the probe and mixes the sample e aspirates a fixed volume of 22 uL of mixed sample independent of the sample volume entered in the software e turns off the waste pump Chapter 1 Introduction 23 24 Sequence Standard Mode High Throughput Mode 6 10 11 moves the probe up over and down into the injection port e injects the sample and boosts it to the cytometer flow cell at high speed e anon selectable fixed boost command transports sample to the flow cell to prime the sample flow path slows down the sample to the required acquisition speed and acquires the required sample volume pushes any remaining sample and the separator bubble through the flow
127. ugh the lines for 30 minutes Allow 60 minutes to complete the monthly cleaning procedure To perform the monthly cleaning you will need DI water and BD FACSClean or a fresh 10 bleach solution 1 part bleach in 9 parts DI water 1 Bypass the cytometer sheath filter e Press the quick disconnectors on both sides of the filter assembly e Remove the filter assembly e Reconnect the two fluidic lines 2 Remove the sheath tank and replace the sheath fluid with BD FACSClean or a freshly prepared 10 bleach solution Replace the tank A To ensure that the 10 bleach solution retains its full germicidal effect prepare a fresh solution daily Chapter 4 Maintenance 111 3 Open the roller clamp on the side of the cytometer for approximately 10 seconds 4 Close the roller clamp 5 Prime the cytometer two to three times to bring the cleaning fluid to the flow cell 6 Place the cytometer in Run mode 7 Choose HTS gt Prime repeat the prime 8 Choose HTS gt Monthly Cleaning The following dialog appears Monthly Clean Place CLEANING tanks on the system Click Continue Cancel 9 Click Continue A dialog appears while cleaning is in progress this can take up to 30 minutes 10 Remove the tank and replace the cleaning solution with DI water Monthly Clean Place RINSING tanks on the system Click Continue Continue Cancel 11 Reinstall the tank A progress dialog appears while ri
128. upplied by BD Biosciences It is the responsibility of the buyer user to ensure that all added electronic files including software and transport media are virus free If the workstation is used for Internet access or purposes other than those specified by BD Biosciences it is the buyer user s responsibility to install and maintain up to date virus protection software BD Biosciences does not make any warranty with respect to the workstation remaining virus free after installation BD Biosciences is not liable for any claims related to or resulting from buyer user s failure to install and maintain virus protection History Revision Date Change Made 338642 10 04 Initial release 640756 3 06 Updated to include BD FACSDiva software version 5 0 and compatibility with BD FACSCanto and BD FACSCanto II flow cytometers 642224 6 07 Updated to include BD FACSDiva software version 6 0 added configuration and performance tracking module operational improvements and increased cytometer specific improvements Contents About This Guide GONVENTIONS APEE ae aie A on BE hs Stet as Sea ee NES Technical Assistante sis saai occu eae case 6 OSs Je SNA ee A a Chapter 1 Introduction HTS Hardware Overview 0 ccc ccc ee eee ce eee eee eees COMPONeNtS o erien i te heed ee e aia RA Oe Eb eee ead ee Cytometer Connections ec cece eee eee eee e eee nee Cytometer Interface Panel 0 0 0 cece ccc eect nen Sainpl Cou
129. ure 4 14 Probe assembly components fitting probe 1 Unscrew the fitting at the top of the probe Figure 4 14 Remove the probe by unscrewing it from the arm Install a new probe A W N Reattach the fitting at the top of the probe Chapter 4 Maintenance 129 Replacing Quick Connector O Rings The sheath and waste lines are attached to the cytometer interface panel and the HTS unit using quick connectors There is an additional waste line attached to the cytometer waste tank using a quick connector Replace the quick connector O rings once a year or whenever a connector is leaking AN All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when changing O rings Wear suitable protective clothing and gloves 1 Disconnect the quick connector 2 Remove the O ring using a flat small screwdriver Figure 4 15 Location of O ring 3 Install a new O ring pushing it into place with your thumbs 4 Reconnect the quick connector Replacing SIT Protector and O Ring BD LSR II Only The SIT protector is a modified sleeve that prevents the sample injection tube from bending during installation of the HTS sample coupler An O ring between the SIT protector and the tube retainer keeps the protector in place Replace the O ring when the SIT protector slips down by itself A All cytometer surfaces that come in contact with biological specimens can tr
130. view after acquisition is complete The printout will contain the plate name plate type name of the view and legend that indicates well status For more information on well status see Plate Layout on this page and Table 2 2 on page 36 BD High Throughput Sampler User s Guide Plate Layout The plate layout is a representation of a multiwell plate The plate layout in the Setup view is where you add samples setup controls and cytometer settings to the plate You can also copy and paste HTS settings rearrange samples add wells assign keywords and set recording rules Once you have set up the plate layout you can print the view to use as a guide when adding samples to the multiwell plate For information on the functions you can perform in Setup view see Setup View Functions on page 43 The plate layout in the Setup view displays the run sequence and status of each well e Wells are numbered according to sample type setup controls first compensation controls second specimens last and then the order assigned e During setup or acquisition wells are colored according to their status See Table 2 1 e After acquisition is complete wells are colored according to acquisition status See Table 2 2 on page 36 The basic layout of each well appears as follows specimen level cytometer specimen control group ID and settings placeholder indicator or first well in group well level cytometer settings placeholder ac
131. words are used to enter information about the wells such as sample type and preparation details For more information refer to the BD FACSDiva Software Reference Manual Chapter 3 Running Samples 77 Preparing for Acquisition 78 Optimizing Cytometer and HTS Settings The default settings provide good starting points to achieve optimal throughput in either mode but the settings should be optimized for your plate type and sample volume 1 Click the Setup tab of the Plate window 2 Click each setup control well view the Cytometer Settings and verify that they are the same as defined in step 1 in Setting Up the Plate on page 67 3 Remove the safety cover on the HTS unit and place the prepared plate on the plate holder AN To prevent damage to the HTS probe always remove the multiwell plate cover before you put the plate on the holder Make sure the plate corresponds to the type selected in the software Orient the plate with well A1 at the back right corner of the stage See Figure 3 14 on page 79 BD High Throughput Sampler User s Guide Figure 3 14 Orienting the plate BD FACSCanto example cytometer interface panel well A1 front of HTS 4 Replace the HTS safety cover 5 BD LSR II Press the RUN button on the cytometer A Do not put the BD LSR II in Standby during HTS acquisition BD FACSDiva software cannot continue the run when the cytometer is in Standby and it can cause damage to the flow cell
132. you try to run the Home command when the cytometer is not in Run mode a software message will remind you that the fluidic system is not ready BD FACSCanto and BD FACSCanto II Ensure the sample coupler is installed and the HTS is turned on 2 Choose HTS gt Home 3 Verify that the HTS properly performs the following homing sequence e The probe moves to the home position if it is not already there e The probe moves up e The plate holder moves left and then slightly forward e The probe moves down e The plate holder moves right to the home position After this sequence the valves initialize priming occurs and the following message appears G HTS Home Complete 4 Click OK to dismiss the message Chapter 4 Maintenance 137 Priming the HTS Prime the HTS unit when you set it up for the first time eg after a depot repair procedure when you notice bubbles in the pump syringe barrel or to clear a clog in the HTS fluidics The Prime command automatically primes both pumps 1 BD LSR II Ensure that the sample coupler is installed and place the cytometer in Run mode To prevent pressure from building in the flow cell the cytometer must be in Run mode before priming begins If you try to run the Prime command when the cytometer is not in Run mode a software message will remind you that the fluidic system is not ready BD FACSCanto and BD FACSCanto II Ensure the sample coupler is installed and the HTS is turned on
133. ype T First well in gro E g 5 ul Name als B Administrator 1 2 3 4 5 6 B Folder_o01 Ali y CN LL Experiment_001 ee Cytometer Setting selected well in hd meee 2 2 2 2 2 current plate or E Global Worksheet Analysis view s 4 obal Worksheets y AE AE JE JL JETA pointer B W 96 Well U bottom AFFF E7 96 Well V bottom Shortcut Menu Right click a selected well at the Analysis view to open the shortcut menu The following commands are available in the shortcut menu Batch Analysis Experiment Layout Copy gt Paste gt Apply Compensation Analysis View Functions Perform the following functions in the Analysis view Chapter 2 BD FACSDiva Software Overview 51 52 Displaying Keyword Results in the Plate Layout If keywords were assigned to the plate clicking on a keyword in the Keywords list toggles the plate layout to show the Keywords Analysis view Each keyword is shown as a different color If a keyword was assigned a range of values a value is displayed in each well Different values of the same keyword are shown in various shades of the keyword color If a well has no value assigned the well is white To revert back to the default Analysis view click the keyword in the Keywords list Use the Keyword Analysis view to display keyword values for all wells that were assigned the selected keyword The Keyword list displays all keywords that were used in the current experiment When you choo

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