Home

BD FACSAria User's Guide

1. pi FF O S flow cell ALG interrogation point OL E nozzle A LM NU O S os 2 g N laser light intercepting flow cell Figure 1 9 Cuvette flow cell The unique flow cell design permits particles to flow through the cuvette at a low velocity allowing longer exposure to laser energy The cuvette is gel coupled to the fluorescent objective lens to transmit the greatest amount of emitted light from the interrogation point to the collection optics see Optics System on page 36 After passing through the cuvette the stream is accelerated as it enters the nozzle tip where the drop drive breaks the stream into droplets for sorting BD FACSAria User s Guide Nozzle The BD FACSAria instrument is provided with two nozzles 70 and 100 um that accommodate a variety of particle sizes Figure 1 10 The nozzle is keyed to a fixed position at the end of the cuvette as shown in Figure 1 9 Because the nozzle is below the interrogation point optical alignment is not affected when the nozzle is changed O ring nozzle inserted ium i in cuvette J Figure 1 10 Nozzle Vv Tip When changing the nozzle do not dislodge the nozzle O ring as you remove or insert the nozzle into the cuvette For instructions see Changing the Nozzle on page 140 A CAUTION The nozzle and its attached stream charging wire conduct electrical charge during sorting To prevent shock do not touch t
2. Setting High Medium Low Custom Sheath Pressure 70 35 20 20 Amplitude 60 50 20 10 Frequency 90 60 30 30 Drop 1 330 330 330 330 Gap 6 6 6 6 Attenuation off on on off Drop Delay 44 00 45 00 29 00 10 00 Chapter 3 Using BD FACSDiVa Software 89 90 Table 3 1 Default Sort Setup values continued Setting High Medium Low Custom Far left voltage 9 45 45 0 Left voltage 40 20 20 0 Right voltage 40 20 20 0 Far right voltage 90 45 45 0 Plate voltage 6 000 5 000 5 000 0 2nd Drop 20 20 10 0 3rd Drop 10 10 5 0 4th Drop 0 0 0 0 Laser Delay blue 0 00 0 00 0 00 0 00 Laser Delay red 36 00 54 00 40 00 82 00 Laser Delay violet 36 00 54 00 40 00 82 00 Area Scaling blue 1 40 1 20 1 40 0 75 Area Scaling red 1 00 0 75 1 00 0 50 Area Scaling violet 0 80 0 35 0 50 0 25 Window Extension 2 00 2 00 2 00 4 00 Sort Layout A Sort Layout is a floating window containing all sorting instructions and controls The Sort Layout designates which device will be used to collect sorted particles and which particles will be sorted into each sort location Up to four sort counters can be displayed in the window to give ongoing status during a sort Only one Sort Layout can be open at a time but you can create several layouts for a single Tube as long as each Sort Layout has a different name Sort Layouts can also be added to Acquisition Templates BD FACSAria
3. El 5 Color Expt B U TENK 001 U TBNK _002 130 BD FACSAria User s Guide 5 On the Acquisition Template create appropriate plots for previewing the data For example create FSC vs SSC FITC vs PE PerCP Cy5 5 vs PE and APC vs APC Cy7 dot plots Mi Tip Ctrl click the Plot tool to keep the tool selected until you create all plots Recording Data Note that if the Save Analysis After Recording preference is enabled in User Preferences a copy of the data displayed in the template will be saved with each recorded Tube If you do not want to save a copy of the data disable the preference 1 Install the first sample tube onto the cytometer 2 Move the Acquisition pointer to the first Tube click Load 3 While data is being acquired draw a region around the lymphocytes set the other plots to show data from the Lymphocyte population 4 Ctrl click the Acquisition pointer to record data 5 When all events have been recorded click Unload and remove the tube from the cytometer 6 Install the next sample tube onto the cytometer move the pointer to the corresponding Tube in the Browser and click Load 7 Preview the data in the Acquisition Template Ctrl click the pointer to record data 8 Repeat steps 5 through 7 until data has been recorded for all tubes 9 Optional Print the Experiment level instrument settings or the Instrument Status report To print the settings right click the instrument
4. lever 3 Turn the nozzle locking lever to the left and pull the nozzle out of the cuvette flow cell Mi Tip To avoid losing the O ring hold the nozzle steady and pull it straight out without tilting it from side to side If you do not see the O ring on the nozzle it could be stuck to the cuvette Use the wooden end of a cotton swab to locate and remove the O ring 140 BD FACSAria User s Guide 8 Transfer the nozzle O ring to the new nozzle or set it aside with the nozzle Carefully insert the new nozzle into the flow cell Make sure an O ring is installed inside the groove at the end of the nozzle Insert the nozzle at a slight downward angle to avoid contact between the O ring and the cuvette Push it gently all the way forward without rocking 1t from side to side Turn the nozzle locking lever clockwise to the 12 00 position and plug the stream charging wire into the nozzle After you start up the instrument turn on the stream and make sure it runs through the nozzle properly Turn on the lasers Note that after changing the nozzle you might need to change the angle of the sort block to re center the stream in the aspirator To do so loosen the adjustment screws on either side of the deflection plates and rotate the sort block see Figure 1 11 on page 32 Tighten the screws when the stream is centered in the aspirator For further assistance see Troubleshooting the Stream on page 194 Setting Up the Breakoff 1
5. Chapter 4 Running Samples 119 Optimizing Signal from the Violet Laser Repeat the steps in Optimizing Signal from the Red Laser on page 118 using plots and parameters appropriate for the violet laser Reusing the Experiment To reuse this Experiment do the following 1 Open the instrument QC Experiment 2 Right click the most recent Specimen and choose Duplicate without Data The three QC Tubes appear under the new Specimen 3 Rename the Specimen with the current date 4 Double click the Blue template to display the plots move the Acquisition pointer to the Blue Tube 5 Continue with the QC procedure e For the blue laser verify the area scaling and adjust the PMTs e For the red and violet lasers verify the laser delay and area scaling and adjust the PMTs 120 BD FACSAria User s Guide Sample Optimization Using Instrument Setup Before you collect data for a stained sample you need to optimize instrument electronics for your sample type Sample optimization consists of five main steps each step is explained in greater detail in the sections that follow It is important that you perform these steps in the order in which they are listed You might need to vary certain steps for different sample types 1 Create an Experiment specify parameters and add compensation Tubes 2 Adjust the FSC and SSC voltages and the FSC threshold 3 Gate the population of interest 4 Adjust fluorescence detectors 5 Calculate co
6. Optical Components The following filters and mirrors are mounted on the BD FACSAria instrument Use these part numbers if you need to order any replacement components Detector Array LP Mirror Replacement Laser BP Filter Part No Intended Dye Octagon 735 334751 488 nm blue laser 780 60 19 62774 18 PE Cy7 655 334752 695 40 19 62774 16 PerCP CyS 5 or PI 675 20 19 62774 02 PerCP alone 595 334753 610 20 19 62774 14 PE Texas Red 556 334754 575 26 19 62774 02 PE or PI 585 42 19 62774 03 Alternative for PE PI when not using PE Texas Red 502 334755 530 30 19 62774 00 FITC 488 10 53 10084 00 SSC Trigon 735 19 62774 18 633 nm red laser 780 60 19 62774 18 APC Cy7 660 20 19 62774 05 APC 212 BD FACSAria User s Guide The following filters and mirrors are provided with the violet laser option Accessory Kit Detector Array LP Mirror Replacement Laser PMT BP Filter Part No Intended Dye Trigon A 502 334755 407 nm violet laser 530 30 19 62774 00 Alexa Fluor 430 B 450 40 334548 Cascade blue Pacific blue Hoechst DAPI Alexa Fluor 405 The instrument is shipped with an accessory kit containing the following items Use these part numbers if you need to order any replacements Item Part No 1 mL microtube holder 333457 12 x 75 mm test tube holder 333456 15 mL centrifuge tube holder 333430 Four way 1 mL collection
7. Use the slider control to adjust the Ampl until the drop breakoff is approximately in the center of the Breakoff window Do not exceed 70 volts If you cannot achieve a drop breakoff at lt 70 volts do the following e Check the flow cell for air bubbles If you see bubbles turn the stream off and back on e Make sure the sheath pressure and drop drive frequency are appropriate Restore the default Sort Setup values if needed e Inspect your nozzle for damage and replace it if necessary Chapter 5 Sorting 141 NOTICE Stream window Verify that the small satellite droplets are merging with the large droplets Satellite merging is largely dependent on nozzle position If the satellites are not merging try to push gently forward on the nozzle Use care not to move the nozzle up or down or side to side You might need to remove and reinstall the nozzle making sure that the nozzle is pushed fully forward before turning the locking lever Input the actual Drop 1 value as the target in the Drop 1 field The actual value appears in the gray background next to the Drop 1 field Once a valid target has been established you do not usually need to reset it unless you change the nozzle or your Sort Setup option NOTICE Ifthe target value causes the Ampl to exceed 70 volts perform the recommendations under step 1 and re input the Drop 1 target value Turn on the Sweet Spot when the drop pattern is stable The Sweet S
8. Y Clean Flow Cell Please wait Click OK when the completion message appears Wait a few minutes and then restart the stream and lasers Chapter 6 Shutdown and Maintenance 171 Prime After Tank Refill Use the Prime After Tank Refill command to prime the fluid lines if a fluidics container was disconnected for refilling 1 Turn off the lasers and the stream if they are on 2 Choose Instrument gt Cleaning Modes gt Prime After Tank Refill 3 Select the checkboxes for the tanks that were refilled click OK i Tank Prime Please select the checkboxes for the tanks that need to be primed M Sheath 7 Diwater Bleach 7 Ethanol Cancel The instrument proceeds with priming the specified tanks The bleach and ethanol tanks are primed before the DI tank is primed A progress message appears while the tanks are being primed click OK to dismiss the message if you would like Y Tank Prime Status G Tank prime in progress Please wait 4 Click OK when you see a message informing you that the tank prime is complete Y Tank Prime Status G Tank prime is complete 172 BD FACSAria User s Guide Long Clean Use the Long Clean command to decontaminate the internal sheath path with bleach or ethanol from the fluid line starting at the outlet of the fluid selection valves in the fluidics cart to the nozzle outlet After decontamination the lines are rinsed with DI water There are three L
9. lower camera window e Clean the strobe lens and upper camera window when smudges appear in the processed digitized image in the Breakoff window or when dark spots appear to interfere with Sweet Spot monitoring You might need to clean these components after a clog or after sheath fluid has leaked or sprayed onto the grounding plate Follow the steps in this section to clean the strobe lens and upper camera window NOTICE Before beginning this procedure turn off the stream and lasers and ensure that the deflection plates are not charged warning light is not illuminated A WARNING A 12 000 volt potential exists between the deflection plates when they are on Contact with the charged plates results in serious electrical shock Do not touch the deflection plates when the plate voltage is on The plates remain energized even when the sort block door is opened 190 BD FACSAria User s Guide 1 Open the sort block door and remove the deflection plates Pull the plates straight out to remove them 2 Remove the nozzle and grounding plate For detailed instructions see Cleaning the Nozzle and Grounding Plate on page 165 You do not need to remove the lever 3 Place 1 2 drops of DI water or ethanol on a cotton swab 4 Right click the Breakoff window and choose Raw Image Visible smudges are more apparent in the raw image view 5 While viewing the image on the Breakoff window insert the swab just below the bottom of t
10. Drop 1 and Gap Drop 1 is defined as the number of pixels from the top of the image to the center of gravity of the first broken off drop A thin gray line on the image is used to identify this drop The number shown to the right of the Drop 1 field at the bottom of the window is the actual pixel location of the gray line The number entered into the Drop 1 field is the user defined Drop 1 target The Gap is defined as the number of pixels from the first discontinuity in the stream to the next stream object generally the first broken off drop A gray line of varying thickness represents the Gap The number shown to the right of the Gap field is the pixel width of the gray line The number entered into the Gap field is a user defined target When the Sweet Spot is turned on the drop drive amplitude Ampl is automatically adjusted to approximately match the target Drop 1 and Gap values The amplitude is initially adjusted in larger increments until Drop 1 is achieved The amplitude is then adjusted in smaller increments until the instrument attains the target Gap The Sweet Spot feature will make adjustments as necessary to maintain the required breakoff conditions throughout the day The Sweet Spot performs two other functions during sorting When sorting if the Drop 1 or Gap are out of range sorting is paused until the values are back within range This ensures that sorting occurs only under the proper breakoff conditions If a more s
11. Recording and Analyzing Data Once you have optimized the instrument electronics for your sample type you are ready to record and analyze data During analysis recorded data is displayed in plots and gates are used to define populations of interest You can use Acquisition Templates to view and optimize data before it is recorded BD FACSDiVa software analyzes the gated data and calculates statistics that you can print or export This section describes how to use BD FACSDiVa software features to record and analyze sample data As an example data will be recorded and analyzed for two samples stained with the following reagents CD4 FITC CD16 CD56 PE CD3 PerCP Cy5 5 CD19 APC CD8 APC Cy7 Two strategies are shown for reusing Analysis objects If you are running the software on an acquisition workstation ie the workstation is connected to the cytometer you can set up the analysis on an Acquisition Template and reuse your analysis strategy for multiple samples in a run Alternatively Analysis objects can be copied to multiple Tubes at a time Setting Up the Acquisition Template This section shows you how to use an Acquisition Template to preview and record data for multiple samples To switch between the Worksheet and Template view click the Template tool on the Worksheet toolbar 5y 1 Create a new Specimen rename the Specimen LWB 2 Create two Tubes under the LWB Specimen rename the Tubes appropriately For example
12. Sample Temperature Use the Sample Temperature command to set the temperature inside the sample injection chamber You can choose from one of the specified values or choose Off to turn off temperature control Figure 3 2 on page 82 Instrument Fluidics Startup Fluidics Shutdown cps Cleaning Modes gt 00 Ing neating unit for sample Sheath Pressure injection chamber Sample Agitation Sample Temperature Y eof Instrument Configuration Instrument Name Enable Test Features Instrument Status Report Figure 3 2 Setting the sample temperature NOTICE The cooling heating unit is designed to maintain the temperature of a sample tube it is not designed to cool or heat the sample Note that it takes approximately 45 minutes to reach the required temperature inside the chamber during which time the chamber must be kept closed To cool or heat the chamber install a sample tube and click Load and then click Acquire to keep the chamber closed and stop running the sample To maintain the temperature do not leave the chamber open for extended periods when changing sample tubes BD FACSAria User s Guide Fluidics Level Indicators BD FACSDiVa software provides fluidics level indicators in the Instrument frame Figure 3 3 The sheath and waste indicators provide an approximate indication of the fluid levels in each tank The DI bleach and ethanol tank indicators appear full until the fluid le
13. When clicked the button changes from a red X to a green checkmark and sheath flows through the cuvette flow cell at the rate that is specified in the Sheath Pressure window See Drop Formation on page 61 for a complete description of the Breakoff window Figure 2 3 Stream control in Breakoff window As a general rule the sheath pressure level is set by choosing a Sort Setup option from the Sort menu rather than by adjusting the Sheath Pressure control Each Sort Setup option is optimized at a preset sheath pressure If you change the sheath pressure a multitude of other values will be affected including the drop drive frequency drop charge levels laser delay area scaling factor and other values For more information see Sort Setup on page 89 Sample Flow Sample is introduced into the cuvette when the Load button is clicked in the Acquisition Controls frame Figure 2 4 on page 48 After Load is clicked the loading port rises to enclose the tube within the sample injection chamber The chamber is automatically pressurized and the chamber pressure forces sample through the sample line into the cuvette flow cell You can stop sample flow when a tube is loaded by clicking the Acquire button Chapter 2 Theory of Operation 47 The sample flow rate is specified using the Flow Rate field in the Acquisition Controls frame You can adjust the Flow Rate from 1 11 which corresponds to approximately 10 120 uL min HE Acquisitio
14. For example use 5 Color Expt or your initials followed by an appropriate identifier 122 BD FACSAria User s Guide 5 Select the Experiment level instrument settings in the Browser click on the Parameters tab in the Inspector and delete any unnecessary parameters Figure 4 6 Click the selection button next to the parameter name s to select the rows press the Delete key or click the Delete button in the Inspector hal Inspector Ea Tube Labels Acq Instr Settings Keywords Parameters Threshold Compensation Ratio Parameter Voltage IICA FSC 260 D m D Dl 55C 300 olewijolo FITC 500 B RB D D PE 500 mai wM olo PercP 0y55 500 BIRD D APC 500 BB D D APC Cy7 500 mimoa E Figure 4 6 Parameters for five color optimization 6 Choose Instrument gt Instrument Setup gt Create Compensation Tubes A Compensation Specimen is added to the Experiment along with a Stained Control Tube for each parameter that was specified in step 5 Worksheets containing the appropriate plots are added for each compensation Tube E P Compensation Specimen 3 laser 9 color 85 Instr Settings H I Unstained Control U FITC Stained Control J PE Stained Control H PerCP Cy5 5 Stained Control UJ APC Stained Control J APC Cy Stained Control 7 Optional Create label specific Tubes if needed Label specific Tubes are not required they are needed only when your experiment con
15. PE Cy7 PI JDS Uniphase HeNe 633 red 11 APC APC Cy7 Air Cooled Point Source Violet 407 violet 10 Alexa Fluor Cascade blue Solid State optional Pacific blue DAPI Hoechst a Measured out of fiber optic cable b Patents PE and APC US 4 520 110 4 859 582 5 055 556 Europe 76 695 Canada 1 179 942 PerCP US 4 876 190 Cy US 5 268 486 5 486 616 5 569 587 5 569 766 5 627 027 PE Cy7 US 4 542 104 APC Cy7 US 5 714 386 BD FACSAria User s Guide Fiber optics direct the laser light in a precise and constant manner onto beam shaping prisms which in turn transmit the laser light to a focusing lens The lens focuses the laser light onto the sample core stream within the cuvette flow cell Figure 1 15 The lasers are positioned on the sample stream for optimal generation of signals Since the optical pathway and sample core stream are fixed optimization is constant from day to day A WARNING Lasers emit intense coherent electromagnetic radiation that can cause irreparable damage to human skin and eyes To prevent retinal burns and possible blindness do not remove laser shielding adjust laser controls or attempt to service the instrument any place where laser warning labels are attached See Precaution Labels on page xvii for the placement of laser warning labels prisms cuvette flow cell L upper camera Figure 1 15 Excitation optics pathway behind laser shielding Chapter 1 BD FACSAria Instrumen
16. Power and Operation 0 c cece cece cent n ence eens 25 Flow Cytometer ose cut te ee eG ede Dd ae ba Pace Sa ae 26 Fluidics Components 0 cece cece eect ence eens 27 Optica ld ee Yee als eee oy 36 Instrument Electronics 1 0 0 cece cee eee ence eee 41 BD FACSAria Workstation 0 0 c cece cece eens 44 iii Chapter 2 Theory of Operation Fluid Movement oo oooooo o Sheath Flow o o o o o o Sample Flow Signal Generation 000 Light Scatter o o Fluorescent Signal Signal Detection o o o oo Detector Arrays Filters ieee aia eee alas Detectors ooooooooomoooo Electronic Processing Pulse Parameters Laser Delay aeran a A Aa SOTIN a Drop Formation Side Stream Formation Drop Charging Conflict Resolution During Sorting Chapter 3 Using BD FACSDiVa Software Workspace Components Instrument Controls Fluidics Controls Fluidics Level Indicators Instrument Configuration Instrument Status Report Acquisition Controls iv BD FACSAria User s Guide 45 46 46 47 49 49 50 51 51 52 56 57 58 59 60 61 65 68 69 77 78 79 80 83 84 85 86 Sorting Controls mulas coria a A A 87 Sort NEME paara a An 88 SESE AAA A ES A A R 89 Sort
17. Recommended Solutions Decrease the Window Extension gt 10 of system event rate Threshold channel too low Increase the threshold channel Event rate too high Decrease the Flow Rate in the Acquisition Controls frame Sample aggregated Filter the sample Sample too concentrated Dilute the sample Fewer events than expected Window Extension set in gated population incorrectly Adjust the Window Extension Refer to the BD FACSDiVa Software User s Guide if needed Laser delay set incorrectly Adjust the laser delay settings See Instrument Quality Control on page 105 Plot zoomed Unzoom the plot or make the gate bigger Events left out of gate When drawing a gate make sure events on the axis are included Increasing threshold results Window Extension too low in decreased Area signal Slightly increase the Window Extension to maximize Area signal A NOTICE Increasing the Window Extension too much results in more electronic aborts or high CVs Area measurement off Area Scaling factor too high scale while the Height measurement is on scale Decrease the Area Scaling factor to move the Area measurement back on scale If necessary adjust Area Scaling to make the Area measurement match the Height measurement 208 BD FACSAria User s Guide Fluidics Cart Troubleshooting Observation No fluid in line during system prime Possible Causes
18. Sorting Beads to Determine the Drop Delay o ooooooo 146 Contents v SOLES aa PO ieee A a PS 149 Setting Up the Experiment 0 cece eee cece eee eens 149 Starting and Monitoring the Sort 0 0 cece eee een eee 151 Setting Up for Sorting Into a Plate or Slide o o o oooooooom oo o 153 Installing the Sorting Hardware 0 c ccc cence cence nee 153 Setting Up the Stream 0 cece eect R 155 Creating a Custom Device 0 cece cee eee ences 158 Chapter 6 Shutdown and Maintenance 161 Daily Shutdown ser crias Pe Bias ias 162 Fluidics Shutdown iscsi deka ode angie shag eae eae 162 External Gleanings 0 54 00 4 4 ets et ode Ra oa A 164 Scheduled Maintenance 0 cece cece tence ences 168 Internal Cleaning ayia yee eee ck wee eed ba beh beeen bebe 168 Purging Ellters css baka aon ane dua kta aaa dado wats 182 Changing Fluid Filters 20 0 cece eee teen eee 183 Changing the Sample Lines 0 0 cece eee cece eee nee 184 Changing Air Filters ooooooooooooomoorronornrooo 187 Unscheduled Maintenance 0 ccc ccc eee enn eee eees 188 Cleaning a Nozzle ccc bhi ee eee Wed oe ag eee eee oe 188 Cleaning the Camera Windows 00 ccc cece ence ence nee 190 Cleaning Optical Filters 2 2 0 cece cee teen ee 192 Chapter 7 Troubleshooting 193 Troubleshooting the Stream 2 1 ccc cette eens 194 Troubleshooting the Breakoff 0 0 0 0 ccc teen eens
19. T B NK_001 and T B NK_002 Chapter 4 Running Samples 129 3 Create an Acquisition Template rename the template Record Data e Tf the Default Acquisition Template is enabled in User Preferences default option the template is already present Expand the Acquisition Templates folder to locate and rename the template e If the Default Acquisition Template preference is disabled create a template by clicking the Acquisition Template tool in the Browser toolbar mj You can create up to ten templates per Experiment 4 Use the Experiment Layout dialog box to define labels and to specify the number of events to record for each Tube Parameter labels are defined in the Experiment Layout view Labels will appear on the plot axes and in all statistics views e Choose Experiment gt Experiment Layout e On the Labels tab enter appropriate labels for the Tube For example enter CDA in the FITC field use the Tab key to move to the next field l 1 Experiment Layout Labels Keywords Acquisition Label cos y E 5 Color Expt PUB FSC ssc FITC PE PerCP Cy5 5 APC U TENK 001 cpa CD16 56 CD3 cD19 FSC 55C FITC PE PerCP Cy5 5 APC T TENK _002 cpa cD16 56_ CD3 CD19 e On the Acquisition tab enter 10 000 events for Tubes 001 and 002 click OK Notice that the Acq tab in the Inspector updates automatically Lit Experiment Layout Labels Keywords Acquisition Events to Record 10000
20. a amp co cn 50 100 150 200 FSC A 1 000 250 Count 020203 Blue 200 FSC A 250 e 1 000 Count 020203 Blue 50 100 150 200 250 SSC A 1 000 020203 Blue 50 100 150 200 FITC A amp Count 250 1 000 Count 020203 Blue 100 PE A 150 200 250 x 1 000 Count 020203 Blue 50 100 160 200 250 PE Texas Red 1 000 020203 Blue Count 50 100 150 200 FITC H 4 250 1 000 Count 020203 Blue 50 100 150 200 PerCP Cy5 5 4 1 000 250 Count 020203 Blue 50 100 150 200 250 PE Cy7 A 1 000 Experiment Name Feb 03 jg SpecimenName 020203 Tube Name Blue Collection Date FSC A FSC A SSC A SSC A FITC AFITC A PE A PE A Population Mean CY Mean cv Feb 2 2003 12 52 32 PM Mean cv Mean CY PE T PE T Mean cv PerC PerC Mean CY XX Fse 94 769 2 4 kK ssc 94 769 24 XX Fitc 94 748 23 XX PE 94 745 23 DJ PE TxRd 94 748 23 X Cy55 94 755 23 XX Cy7 94 748 2 3 96 512 96 519 96 491 96 487 96 491 96 500 96 487 6 3 6 3 6 2 6 2 6 2 6 2 6 2 98 101 98 138 97 343 97 339 97 339 97 345 97 337 Figure 4 4 Blue laser data example 9 1 9 3 27 27 2 8 2 7 2 8 99 844 99 880 99 115 99 112 99 113 99 119 99 111 8 6 8 8 2 5 2 5 2 5 2 5 2 5 97 774 97 811 96 997 96 994 96 999 97 005 96 997 Chapter 4
21. and the stream See Starting Up the Instrument on page 100 When you turn on the stream make sure it flows smoothly from the nozzle into the center of the waste aspirator If you need help troubleshooting the stream turn it off and go to Troubleshooting the Stream on page 194 Choose a new Sort Setup option if needed To change your current Sort Setup mode choose High Medium or Low from the Sort gt Sort Setup menu e High is designed for high speed sorting with the 70 um nozzle e Medium is designed for sorting cell lines or fragile cells with the 70 um nozzle e Low is designed for single cell sorting ACDU sorting or sorting large or fragile cells with the 100 um nozzle For more information see Sort Setup on page 89 Adjust the Ampl control to set the required drop breakoff Adjust the Drop 1 value if needed to match the generated value For more information see Setting Up the Breakoff on page 141 If you need help troubleshooting the drop image see Sorting Troubleshooting on page 199 138 BD FACSAria User s Guide 10 11 Turn on the Sweet Spot when the drop pattern is stable Check the laser delay and the area scaling factor for your sheath pressure See Instrument Quality Control on page 105 Perform sample optimization for the sample to be sorted See Sample Optimization Using Instrument Setup on page 121 Install the required collection device and set up the side streams See Setting Up for Bulk
22. laser Optical upgrades are available to fill the remaining PMT slots for a two or three laser system A fully equipped system can detect up to 13 fluorescent parameters and two scatter parameters at a time trigons blue laser signal y N violet laser red laser octagon signal signal Figure 1 16 Fully loaded detector arrays At installation the octagon and trigon arrays are set up with the filter and mirror combinations shown in Table 1 1 on page 40 You can rearrange these configurations according to the type of fluorochromes in your experiment See Application Options on page 220 for more information Chapter 1 BD FACSAria Instrument Components 39 40 Table 1 1 Default setup for detector arrays Detector Array PMT LP Mirror BP Filter Intended Dye Laser Octagon A 735 780 60 PE Cy7 488 nm blue laser 655 695 40 PerCP CyS 5 or PI 675 20 PerCP alone 595 610 20 PE Texas Red D 556 575 26 PEor PI 585 42 Alternative for PE PI when not using PE Texas Red E 502 530 30 FITC F 488 10 Side scatter SSC Trigon A 735 780 60 APC Cy7 633 nm red laser B 660 20 APC Trigon A 502 530 30 Alexa Fluor 430 407 nm violet laser on B 450 40 Cascade blue Pacific blue DAPI Hoechst Alexa Fluor 405 Stream Viewing Optics The BD FACSAria instrument is equipped with additional optical components that are used to view the stream Figure 1 17 on page 41 e The upper camera gen
23. 198 Sorting Troubleshooting 0 ccc ununura anerer 199 Acquisition Troubleshooting 0 00 cece cece eee n eee nes 203 Fluidics Cart Troubleshooting 0 0000 cece cece een eens 209 Electronics Troubleshooting 0 00 eee nee n eens 210 vi BD FACSAria User s Guide Appendix A Supplies and Options 211 Instiument Supplies dat a wee A aa 212 Optical Components 0 0 cece eee een eee eens 212 Accessory Kit asso taaan 20s dese debe Sa 8e 2 EOS a 213 Other Replacement Parts muro rs ohh ee ee ek cee 215 Las r Specifications LA ae oa eR ew Na hee a bs 215 Consumables a 216 Instrument Setup Particles 0 0 cece cece eens 216 Res Bitte Se Bee Rae ORR EE ees ete a e de i 217 Labware 03 oi ted on LSA SS E bee A a 218 Instrument Options 0 0 eee eee eee eee ee eee E 219 Application Options 0 0 0 cece een eens 220 TEwo Laser System dt 220 Three Laser System moroi 0 eect ec ERO eee nes 221 Custom Configuration 0 ccc cnet nent enn enes 222 Changing Filters oooooooooooonmorrrrnrrrrnnno 223 Defining a New Configuration 0 00 0 ccc eee een eens 224 Index 227 Contents vil viii BD FACSAria User s Guide About This Guide This user s guide contains the instructions necessary to operate and maintain your BD FACSAria flow cytometer Because many instrument functions are controlled by BD FACSDiVa software this guide also contains basic so
24. 5 E9 PE Cy7 8 474 8 463 8 468 8 387 8 389 8 388 8 383 8 385 84 7 99 9 99 9 99 0 99 0 99 0 98 9 98 9 Events Parent Total 10 000 100 0 84 7 84 6 84 7 83 9 83 9 83 9 83 8 83 9 5 Edit the Statistics view to show only the named populations Right click the Statistics view and choose Edit Statistics View Click on the Populations tab and deselect the checkbox for the All Events population Chapter 4 Running Samples 115 6 Optional Print the instrument settings or the Instrument Status report The instrument settings printout contains a record of the PMT voltages and compensation settings the Instrument Status report includes the sheath pressure and flow rate lasers used Laser Delay and Area Scaling settings and the Window Extension in addition to the instrument settings The Laser Delay and Area Scaling settings for a recorded Tube can also be viewed on the Tube tab of the Inspector hal Inspector xj Tube Labels Acq Instr Settings Keywords Name Blue Total of Events foooo oo Acquisition Date eros Acquisition Stat 3 53 26 AM Acquisition End s359aM SCS Cytometer Name Facsaia oo Cytometer Serial fokker Laser Delay Blue 0 00 Red 39 00 Violet35 00 Area Scaling Blue 1 55 Red 1 15 Violet1 35 Window exensionf300 7 Optional Print the data on the Blue template See Figure 4 4 on page 117 for an example 116 BD FACSAria User s Guide 020203 Blue s 8 3
25. 60 DF filter in front of it which transmits light of 750 810 nm Thus the only wavelengths that will reach the A detector are those between 750 and 810 nm For optimal detection of fluorescent light a bandpass filter must always be installed in front of each detector For a list of the bandpass filters used in the detector arrays see Table 1 1 on page 40 Chapter 2 Theory of Operation 55 56 Detectors Detectors within each detector array convert light signals into electrical signals that can be processed by the electronics system There are two types of signal detectors in the BD FACSAria flow cytometer the photodiode and photomultiplier tubes PMTs The photodiode is less sensitive to light signals than the PMTs thus is used to detect the stronger FSC signal The photodiode detects FSC light from the blue laser and is stored outside the detector arrays PMTs are used to detect the weaker signals generated by SSC and all fluorescence channels The octagon can hold up to eight PMTs each trigon can contain up to three PMTs Each PMT detects only one fluorochrome at a time In BD FACSDIiVa software the Instrument Configuration dialog box lets you define which fluorochromes or cell parameters will be measured at each PMT detector If more than one fluorochrome is measured using the same PMT you can add additional parameters to your configuration and choose the appropriate fluorochrome within your software Experiment See Defining
26. Bandpass BP filters transmit a relatively narrow range or band of light Bandpass filters are typically designated by two numbers The first number indicates the center wavelength and the second refers to the width of the band of light that is passed For example a 500 50 BP filter transmits light that is centered at 500 nm and has a total bandwidth of 50 nm Therefore this filter transmits light between 475 and 525 nm Figure 2 9 100 bandpass 460 500 540 480 520 i 2 2 50 g 5 a 0 450 500 550 wavelength nm Figure 2 9 Light transmittance by bandpass filters Discriminating DF filters have the same general function they transmit a relatively narrow band of light The principal difference between BP and DF filters is their construction DF filters have more cavities or layers of optical coatings resulting in a steeper transmission curve than the curve for a BP filter This steep slope means that a DF filter is better at blocking light outside the rated bandwidth of the filter See Figure 2 10 on page 55 BD FACSAria User s Guide 100 we BP 500 50 filter DF 500 50 filter 50 transmission 450 500 550 wavelength nm Figure 2 10 Bandpass BP vs discriminating DF filters In the detector arrays DF filters block high intensity laser light and filter the remaining light to ensure that only the required wavelengths reach their intended detector For example in the octagon array PMT A has a 780
27. Sorting on page 143 or Setting Up for Sorting Into a Plate or Slide on page 153 Calculate the drop delay See Determining the Drop Delay on page 145 Run the sample to be sorted and use gating tools and subsetting methods to define the population s of interest Examples of gating analysis can be found in Analyzing Data on page 132 and in the BD FACSDiVa Software User s Guide Define a Sort Layout for the Tube containing the defined sort populations and proceed with sorting See Sorting on page 149 Chapter 5 Sorting 139 Changing the Nozzle As a general guideline the nozzle opening should be at least three times the diameter of the particle to be sorted Two sizes of nozzles are provided with your instrument 70 and 100 um nozzles The size is marked on the nozzle b WARNING Any instrument surface that comes in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling sorting hardware Wear suitable protective clothing and gloves A CAUTION The nozzle and the stream charging wire conduct electrical charge during sorting To prevent shock do not touch the nozzle or the stream charging wire when the voltage warning light is illuminated or any time during sorting To change the nozzle do the following 1 Turn off the lasers and the stream if they are on 2 Pull the stream charging wire out of the nozzle stream charging wire
28. User s Guide Sort Layouts are available for up to nine default collection devices additional custom devices can be defined See Creating a Custom Device on page 158 24 Well Falcon 48 Well Falcon 96 Well Falcon 384 Well Falcon default collection devices Slide Frosted End Slide Standard Examples of Sort Layouts for different devices are shown in the following figures instructions for setting up the Sort Layout can be found in Setting Up a Sort Layout on page 92 Device Precision Target Events Save Conflicts collection S Tuve y Purity z device Leii sort location field for far right tube Sort Rate sort Conf Cnt NA NA NA NA counters Conf Rate T NA NA NA Efficiency NA NA NA sorting WM Sort WM Pause ajz ss View Counters controls Device Precision Target Events Save Conflicts fas Well Costar y sinaie Cell y mj 4 5 6 sort location field for individual well Sort Rate Confl Cnt Confl Rate Efficiency Bl Sort mi Pause Aj View Counters Figure 3 5 Sort Layout for collection tubes top and for 48 well plate bottom Chapter 3 Using BD FACSDiVa Software 91 92 Sort Sample Sort Layout xj Device Precision Target Events Save Conflicts feige Frosted End ws Single Cell v y s
29. a Yield Mask of zero is recommended Yield Masks cannot be used in conjunction with Phase Masks Thus when the Yield Mask is greater than zero the Phase Mask automatically reverts to zero BD FACSAria User s Guide Purity Mask The Purity Mask setting defines how close in 1 32 drop increments a contaminating drop can be located before ignoring the drop being interrogated For example when the Purity Mask is set to 16 the drop being interrogated will not be sorted if a non target particle falls within the first or last 8 32 of the leading or trailing drop In the following example a non target particle falls within the first 8 32 so the interrogated drop will not be sorted Figure 2 20 trailing drop drop being interrogated leading drop xx w Purity Mask noti sortea Purity Mask non target particle Figure 2 20 Non target particle within a Purity Mask of 16 If the Purity Mask were set to 8 for the same target particle the non target particle would fall outside of the Purity Mask so the interrogated drop would be sorted See Figure 2 21 trailing drop drop being interrogated leading drop og Vy Purity Mask sorted Purity Mask non target particle Figure 2 21 Non target particle outside a Purity Mask of 8 NOTICE With any Purity Mask greater than zero the drop being interrogated must be free of co
30. a New Configuration on page 224 for more information Lit instrument Configuration Current Configuration 3 laser color Side Scatter Parameter Blue F y Parameter Laser Detector FSC Blue 2 laser 7 color ao O a ssc Blue F FITC Blue E PE Blue D PE Texas Red Blue e PerCP Cy5 5 Blue B PE Cy Blue A IAPC Red la APC Cy7 Red A Violett Violet JB Violet2 Violet lA le Add Configuration Delete Configuration Set Configuration Add Parameter pelete Parameter Figure 2 11 Default BD FACSAria instrument configurations BD FACSAria User s Guide Electronic Processing As cells or other particles pass through the focused laser beams they scatter the laser light and can emit fluorescence Because each laser beam is focused on a small spot and particles move rapidly through the flow cell the scatter or fluorescence emission has a very brief duration only a few microseconds The PMTs convert this brief flash of light into an electrical signal called a pulse Figure 2 12 1 A pulse begins when a particle enters the laser beam At this point both the beam intensity and signal intensity are low 2 The pulse reaches a maximum intensity or height when the particle reaches the middle of the beam where the beam and signal intensity are the brightest The peak intensity or height of the pulse is measured at this point 3 As the particle leaves the beam the pul
31. actual Drop 1 value for the target Remember to repeat the drop delay setup each time the target value is changed Nozzle clogged or inserted improperly Turn off the stream Remove the nozzle and clean it Reinsert the nozzle at a slight downward angle to prevent loss or movement of the O ring Make sure the nozzle is completely seated against the back wall of the cuvette flow cell Dirty strobe lens or upper camera window Clean the lens and the window as described in Cleaning the Camera Windows on page 190 Center stream image dim Stream not intercepting or not visible in Side diode laser Stream window Adjust the micrometer dial on the laser to make the stream intercept as bright as possible See Figure 1 17 on page 41 Center stream off center Voltage Center too low or too when plate voltage turned high Adjust the Voltage Center slider to put the center stream back to center on Saline spray on deflection plate or in sort block s Clean the deflection plates and the area around them Arcing between deflection Plates too close together at the Adjust the plates so there is a gap of plates top approximately 0 28 in 0 7 cm between them at the top Salt bridge Clean and dry the deflection plates and the area around and behind the plates Chapter 7 Troubleshooting 199 Sorting Troubleshooting continued Observation Possible Causes ACDU sorting failure Insufficient stre
32. cable power cord air input clear connections sheath input blue for temperature control option aspirated fluid output yellow fluid output orange connection Figure 1 4 Fluidics cart power and fluid line connectors To connect the fluidics cart to an external air source open the shutter above the Communication port on the fluidics cart flip the Auxiliary Air Supply switch to On and attach the external air line to the air input connector supply switch connector NOTICE The external air supply should provide 80 100 psi 24 BD FACSAria User s Guide Power and Operation Power to the fluidics cart is supplied by the cytometer the cart s fluid pumps are activated when the cytometer main power switch is turned on See Power Panel on page 42 A NOTICE Power to the fluidics cart is supplied and controlled through the flow cytometer The fluidics cart voltage settings have been configured to match the supply voltage by your service engineer To properly operate the fluidics cart plug the fluidics cart power cord only into the power receptacle on the instrument Figure 1 4 on page 24 Do not plug the power cord directly into a wall socket Do not change the input voltage selection jumpers on the fluidics cart fuse holder A NOTICE When the stream is on air pressure fluctuates between 80 100 psi Figure 1 5 A pressure reading of less than 80 psi or greater than 100 psi indicates that the fluidic
33. chamber door if needed The sort block door must be closed in order to open the collection chamber door Remove the tube holder if one is installed and tilt the splash shield to insert it into the slotted fittings below the sort aspirator drawer Push the splash shield all the way in F splash shield Figure 5 3 Installing the splash shield Chapter 5 Sorting 153 2 Click the Access Stage button to bring the ACDU stage to the front e Open an Experiment if one is not already open and create a Sort Layout for any of the Tubes e Inthe Sort Layout view click the Access Stage button 4 to move the stage to the front of the sort collection chamber 3 Install the required collection device on the stage e If you are sorting into a plate install the plate with well A1 toward the front of the stage e If you are sorting onto a slide install the slide adapter tray with the printed side up If your slide has a frosted end place the frosted end to the right Figure 5 4 ACDU stage set up with plate left and slide right Sorting starts on the front left corner of the device A1 location and proceeds from front to back and then from the left to the right side of the device sorting in a serpentine motion Thus for a plate sorting proceeds from well A1 A12 B12 B1 C1 C12 and so on When sorting onto a slide sorting proceeds in rows across the short end of the slide and in columns along the long end o
34. checkbox if needed UU Bb WU N Click Close The new mode is added to the Precision Mode drop down menu To delete a mode choose it from the drop down menu and then click Delete Chapter 2 Theory of Operation 75 76 BD FACSAria User s Guide 3 Using BD FACSDiVa Software All BD FACSAria instrument functions are controlled using BD FACSDiVa software This chapter provides a general overview of the workspace components and describes instrument controls that are unique to the BD FACSAria instrument For an in depth description of software components not described in this chapter refer to the BD FACSDiVa Software User s Guide The following topics are covered in this chapter e Workspace Components on page 78 e Instrument Controls on page 79 e Sorting Controls on page 87 77 Workspace Components When you start BD FACSDiVa software the workspace appears Figure 3 1 Frames containing the main application components are displayed within the workspace by clicking the corresponding buttons in the Workspace toolbar in the figure Frames shown in Figure 3 1 are numbered 2 7 Two additional frames can be displayed by clicking the Sorting button in the Workspace toolbar see Sorting Controls on page 87 for a description Lit BD Symphony Software lej x File Edit View Experiment Populations Worksheet Instrument Sort Help HA BRERA l N Browser Fr Worksheet xi B R REARS Esee SEAS aiae E She
35. cuvette flow cell Chapter 7 Troubleshooting 197 Troubleshooting the Breakoff Use the following examples to help troubleshoot problems with the breakoff image E lt a Stream mj Sweet Spot a stream 12 Sweet Spot ii stream pa Sweet Spot m Stream bal Sweet Spot i mM Stream Dal Sweet Spot A mM Stream Dal Sweet Spot o v gt a 6 t 0 o E 0 a 5 oO pd e a amp bli y lt x 0 8 o E 0 K Jampl 86S lAmpl 23123 Jampi 4465 Jampi 80 0 4 Freq 90 0 Freq 90 0 St E Freq 90 0 Freq 90 0 IDrop1 306 71 Drop1 256 138 Drop 1 306 499 Drop 1 306 485 IDrop1 306 0 joo a 6 30 Gap 6 5 Gap 6 18 poe E Go pens Wo 6 0 Normal stream 2 Nozzle Nozzle Partial clog Wet or dirty Attenuation on image 2 Y inserted inserted strobe lens at wrong wus A es improperly improperly pressure Remove the Remove the Remove the Clean the lens Turn off 3 nozzle and nozzle and nozzle clean as described in attenuation in g 2 reinsert it reinsert it it and then Cleaning the the Side ES reinsert it Camera Stream 5 E i gs Windows on window Y ZA page 190 198 BD FACSAria User s Guide Sorting Troubleshooting Observation Possible Causes Unstable breakoff while Target Drop 1 value out of Sweet Spot engaged range for drop spacing Recommended Solutions Use an
36. details of recent system performance For instrument support from within the US call 877 232 8995 prompt 2 2 For support from within Canada call 888 259 0187 About This Guide xi Customers outside the US and Canada contact your local BD representative or distributor xii BD FACSAria User s Guide Safety and Limitations The BD FACSAria flow cytometer is equipped with safety features for your protection Operate the instrument only as directed in the user s guide Keep this safety information available for future reference and for new users Laser Safety Lasers or laser systems emit intense coherent electromagnetic radiation that has the potential of causing irreparable damage to human skin and eyes The main hazard of laser radiation is direct or indirect exposure of the eye to thermal radiation from the visible and near infrared spectral regions 325 1 400 nm Direct eye contact can cause corneal burns retinal burns or both and possible blindness There are other potentially serious hazards in other spectral regions Excessive ultraviolet exposure produces an intolerance to light photophobia accompanied by redness a tearing discharge from the mucous membrane lining the inner surface of the eyelid conjunctiva shedding of the corneal cell layer surface exfoliation and stromal haze These symptoms are associated with photokeratitis otherwise known as snow blindness or welder s flash which results from ra
37. engineer during installation Limitations This instrument is for Research Use Only Not for use in diagnostic or therapeutic procedures Safety and Limitations xix xx BD FACSAria User s Guide BD FACSAria Instrument Components The BD FACSAria flow cytometer is the first fixed alignment benchtop high speed cell sorter The instrument can be operated at varied pressures and can acquire up to 70 000 events per second With its fixed optics design and digital electronics the BD FACSAria flow cytometer enables multicolor analysis of up to 13 fluorescent markers and two scatter parameters at a time The BD FACSAria system consists of three major components a fluidics cart a benchtop flow cytometer and the BD FACSAria workstation see Figure 1 1 on page 22 Nearly all instrument functions are operated from within BD FACSDiVa software For a description of the BD FACSAria system components see the following sections For technical information about how the instrument works see Chapter 2 e Fluidics Cart on page 22 e Flow Cytometer on page 26 e BD FACSAria Workstation on page 44 21 flow cytometer fluidics cart workstation Figure 1 1 BD FACSAria instrument components Fluidics Cart A separate fluidics cart supplies sheath and cleaning fluids and collects waste from the instrument Figure 1 2 The self contained fluidics cart supplies the required air pressure and vacuum which eliminates the need for
38. for the fluorescent populations Figure 4 5 on page 119 118 BD FACSAria User s Guide 10 11 IET METE E Status Parameters Threshold Status Parameters Threshold Compensation Ratio Laser Compensation Ratio Laser Name Delay Area Scaling Name Delay Area Scaling Blue 0 00 1 65 Blue 0 00 1 65 0 95 Red J 37 60 ait 0 95 0 76 Violet 32 80 0 76 Window Extension 2 000 44 Window Extension 0 000 4 020203 Red 020203 Red I l m 539 ary o o o o 50 100 150 200 250 50 100 150 200 250 APC A x 1 000 APC A x 1 000 Figure 4 5 Second laser delay before left and after right adjustment e Reset the Window Extension to the appropriate setting typically 2 A larger Window Extension allows more flexibility for capturing pulses Verify area scaling for the red laser and adjust it if needed Adjust area scaling until the APC A intensity matches that in the APC H plot To adjust the value select the current value and enter a new value View the result of your change in the histogram plots See Figure 4 3 on page 114 for an example Optimize the PMT voltage for each fluorescent parameter Adjust the voltage to achieve a signal of approximately 100 x 107 in the corresponding fluorescence histogram Record and analyze data for the red laser Repeat the steps listed under Recording and Analyzing Data on page 115 substituting appropriate parameters for the parameters listed
39. illuminated whenever the plate voltage is on Figure 1 13 on page 34 A WARNING A 12 000 volt potential exists between the deflection plates when they are on Contact with the charged plates results in serious electrical shock Do not touch the deflection plates when the voltage warning light is illuminated or when the software indicates that the plate voltage is on The plates remain energized even when the sort block door is opened Aspirator Drawer The aspirator drawer keeps the sort collection tubes covered until sorting begins Figure 1 12 You can open and close the drawer using a control in the Sort Layout view see Using Sorting Controls on page 94 The drawer closes automatically to protect your sort collection tubes when a clog is detected during sorting A CAUTION To avoid pinching your hands or fingers in the drawer keep your hands away from the sort block during sorting Figure 1 12 Aspirator drawer closed left vs open right Chapter 1 BD FACSAria Instrument Components 33 34 Aerosol Containment During sample acquisition and sorting the sort block door should be kept closed to help contain potential aerosols Figure 1 13 WARNING Cell sorters that use droplet generation methods like the BD FACSAria can produce aerosols around the sample stream Inhalation or contact with aerosols exposes you to biologically transmissible diseases When acquiring biohazardous samples follow universal precautions at a
40. instrument and do not need user adjustment A NOTICE The flow cell access door is equipped with a shutter mechanism that shuts off the laser light when the door is opened To ensure there is no interruption to data acquisition do not open the door while sorting or recording 26 BD FACSAria User s Guide See the following sections for more information about the flow cytometer e Fluidics Components on page 27 e Optics System on page 36 e Instrument Electronics on page 41 Fluidics Components When the fluidics system is activated sheath fluid is pumped from the fluidics cart into a plenum reservoir where the fluidics system is pressurized From the reservoir fluid is pumped into the cuvette flow cell where hydrodynamic focusing forces particles from the sample injection chamber through the cuvette in a single file stream Within the cuvette flow cell laser light is focused on the sample core stream Fluorescent molecules excited by the different laser wavelengths are detected by the optics and analyzed by the electronics Particles are then either transported to waste reservoirs via the waste aspirator or sorted into a receptacle within the sort collection chamber The following fluidics components are described in this section For more information about fluidics see Fluid Movement on page 46 e Plenum Reservoir on page 28 e Sample Injection Chamber on page 29 e Cuvette Flow Cell on page 30 e Nozzle on page 31 e Sort Block on
41. keep the system free from salt buildup wipe down all instrument surfaces that have been exposed to sheath fluid Clean surfaces with a cloth dampened with a 10 bleach solution followed by DI water b WARNING All instrument surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when cleaning instrument surfaces Wear suitable protective clothing and gloves A WARNING To prevent shock turn off the plate voltage before cleaning on or around the deflection plates To prevent arcing sparking make sure the plates are completely dry before you turn the plate voltage back on The following surfaces should be inspected and cleaned when necessary e inside the sort block e deflection plates e grounding plate see the following section 164 BD FACSAria User s Guide Cleaning the Nozzle and Grounding Plate Tf sheath fluids leaks or sprays onto the grounding plate within the cuvette flow cell you will need to remove the plate and clean and dry it thoroughly before you continue operating the instrument Perform this procedure when you observe no deflection or insufficient deflection during sorting or anytime the nozzle gets wet b WARNING Any instrument surface that comes in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling sorting hardware Wear suitable protective clothing and gloves 1 Turn off the
42. laser so the red laser signals always have a negative Delay value Figure 2 14 on page 60 Chapter 2 Theory of Operation 59 Instrument Lx Status Pi io Laser Name Delay Area Scaling Blue 0 00 1 40 Red 38 80 1 05 Violet 35 20 0 70 Window Extension 2 000 St Figure 2 14 Delay values in Instrument frame Sorting 60 During sorting drop drive energy is applied to the stream to break it into highly uniform droplets Droplets detach from the stream a few millimeters downstream from the nozzle The time between when a particle intercepts the laser and when it reaches the droplet breakoff point is determined using BD FACS Accudrop technology see Setting the Drop Delay on page 67 When a particle is detected that meets the predefined sorting criteria an electrical charge is applied to the stream just as the droplet containing that particle breaks off from the stream Once broken off from the stream the droplet now surrounded by air still retains its charge The charged droplet passes by two strongly charged deflection plates Electrostatic attraction and repulsion cause each charged droplet to be deflected to the left or right depending on the droplet s charge polarity Uncharged droplets are not affected by the electric field and pass down the center to the waste aspirator See Figure 2 15 on page 61 The following sections describe how the BD FACSAria instrument creates and ch
43. of the previous drop from 100 to 100 Adjusts the phase between drop generation and charging of the droplets from 0 360 degrees The selected value is sent to both the drop charging electrode and the drop strobe In general the Phase never needs adjusting you can keep the default value of zero BD FACSAria User s Guide Setting the Drop Delay The BD FACSAria instrument includes integrated Accudrop technology to assist in setting an accurate drop delay value Accudrop components consist of the following e a diode laser mounted to the left of the sort block e a camera that provides an image of the side streams e an emission filter for viewing the fluorescence from Accudrop beads The emission filter is installed in front of the lower camera and can be moved in and out by clicking the Optical Filter control When the button is yellow OJ the filter is out This position is used to view the center and side streams Click the button to move the filter in front of the camera when you are determining the drop delay the button changes to red 4 To determine the drop delay the streams are illuminated by the diode laser just below the point of deflection Specialized fluorescent particles Accudrop beads can be viewed in the center and left side streams as the delay is adjusted The best delay yields the most particles in the left steam and the fewest in the center stream See Determining the Drop Delay on page 145 for more informat
44. page 32 e Sort Collection Chamber on page 35 Chapter 1 BD FACSAria Instrument Components 27 28 Plenum Reservoir Sheath fluid is pumped from the fluidics cart into a plenum reservoir inside the side door of the BD FACSAria flow cytometer Figure 1 7 The plenum reservoir holds the sheath fluid until it is pumped into the cuvette flow cell It eliminates pressure fluctuations by maintaining fluid levels and filtering out pump pulsation As a result the fluidics pressure does not vary with the level of fluid in the sheath containers Figure 1 7 Plenum reservoir Note that the plenum tanks can be unscrewed and removed for autoclaving or cleaning if needed See Cleaning the Plenum Reservoirs on page 175 for more information BD FACSAria User s Guide Sample Injection Chamber The sample injection chamber is where sample is introduced into the flow cytometer During acquisition the chamber is pressurized to force sample toward the cuvette flow cell Samples can be agitated and temperature controlled within the sample injection chamber using controls in the software see Fluidics Controls on page 80 You can view the amount of fluid remaining in your sample tube by pressing the chamber light button shown in Figure 1 8 NOTICE Do not use the chamber light for long periods with samples stained with light sensitive reagents sample injection chamber chamber light tube holder loading port 1 microti Figure 1 8 Samp
45. screws See Figure 1 11 on page 32 Nozzle inserted improperly Turn off the stream Remove the nozzle and ensure that the O ring is in place Reinsert the nozzle at a slight downward angle to prevent loss or movement of the O ring Make sure the nozzle is completely seated against the back wall of the cuvette flow cell Clogged or damaged nozzle Turn off the stream remove the nozzle and examine the nozzle tip under a microscope e If debris is visible clean the nozzle See Cleaning a Nozzle on page 188 e If the nozzle appears damaged replace it See Changing the Nozzle on page 140 194 BD FACSAria User s Guide Troubleshooting the Stream continued Observation Possible Causes No stream or dripping Grounding plate inserted Recommended Solutions Make sure the grounding plate is stream improperly inserted with the notched end toward you See Cleaning the Nozzle and Grounding Plate on page 165 Clogged or damaged nozzle Turn off the stream remove the nozzle and examine the nozzle tip under a microscope e If debris is visible clean the nozzle See Cleaning a Nozzle on page 188 e If the nozzle appears damaged replace it See Changing the Nozzle on page 140 Stream control disabled Plenum not full Wait for the plenum to fill or no stream when Stream control clicked Air lock in filter Prime the system with the corresponding fluid Tf the control is still
46. the Tube s worksheet Saving the Analysis Since the Analysis objects were created on an Acquisition Template the analysis will not be saved with each Tube If you want to save a copy of the analysis with any Tube do the following 1 Expand the T B NK Analysis Template in the Browser 2 Right click on the Analysis object and choose Copy 2 48 5 Color Expt 8S Instr Settings 3 Acquisition Templates Record Data 3 TIBINK Analysis object within template 3 Click the Template tool on the Worksheet toolbar BJ to switch to the Worksheet view Chapter 4 Running Samples 135 4 Create a new worksheet for the destination Tube rename the worksheet appropriately 5 Collapse the Tube in the Browser right click the Tube icon and choose Paste The elements on the template are copied to the new worksheet You can view the analysis by double clicking the Tube in the Browser T Tip Apply the analysis to multiple Tubes by selecting more than one Tube before you choose Paste Enable the Tube specific worksheet user preference to automatically create a new worksheet for the pasted Analysis objects Note that you can automatically save a copy of the analysis with each Tube by enabling the Save Analysis After Recording preference before you record Tube data In this case the analysis plots are placed on the worksheet that is open at the time of recording To control where the plots are placed create a new worksheet before
47. the new filter and connect the quick disconnect couplings Mi Tip Write today s date on the filter so you will know when to replace it 3 Open the bleeder valve on top of the filter and leave it open until fluid seeps out through the valve 4 Close the valve Chapter 6 Shutdown and Maintenance 183 Changing the Sample Lines The primary sample line between the sample injection chamber and the HPLC valve should be changed every 4 6 months or when decreased event rates indicate that the sample line might be clogged The secondary sample line between the HPLC valve and the cuvette flow cell needs changing only when it is kinked or clogged To withstand the high pressures generated by the BD FACSAria flow cytometer the sample lines are attached at each end using a two piece compression fitting where a cone shaped ferrule is compressed onto the tubing as the connecting nut is tightened To replace the tubing you will need a length of replacement tubing from the accessory kit The 12 in length of tubing is used for the primary sample line the 7 in length is used for the secondary line Figure 6 4 The ferrules and connecting nuts can be reused when the tubing is replaced b WARNING All biological specimens and materials coming into contact with them can transmit potentially fatal disease Handle used tubing and fittings as if capable of transmitting infection Wear suitable protective clothing and gloves secondary sample line
48. to diagnose and adjust certain instrument settings Other menu commands Instrument Name Instrument Setup are described in the BD FACSDiVa Software User s Guide Additional instrument controls are located in the Acquisition Controls frame see Acquisition Controls on page 86 Chapter 3 Using BD FACSDiVa Software 79 80 Fluidics Controls Fluidics control of the BD FACSAria instrument is completely automated by BD FACSDiVa software The software contains pre programmed fluidics protocols that are activated by choosing the corresponding menu command from the Instrument menu In addition fluidics level indicators are available in the Instrument frame see Fluidics Level Indicators on page 83 Fluidics Startup The Fluidics Startup procedure verifies waste and sheath levels and primes the fluidics system with sheath solution See Starting Up the Instrument on page 100 for more information Fluidics Shutdown The Fluidics Shutdown procedure removes sheath from the lines and plenum and replaces it with DI water and cleans the cuvette with a cleaning solution See Fluidics Shutdown on page 162 for more details Cleaning Modes BD FACSDiVa software contains pre programmed cleaning modes that are activated by choosing the corresponding menu command from the Instrument gt Cleaning Modes menu See Internal Cleaning on page 168 for more information Fluidics Startup Fluidics Shutdown Sample Line Backflush Shea
49. toolbar mp 2 Select the template Template 1 in the Browser and rename it Blue 3 Select the Blue template in the Browser and create appropriate plots for the blue laser NOTICE Make sure the Worksheet frame is displaying the template view before you create any plots If necessary click the Template button in the Worksheet toolbar 3 to switch to the Template view For example create the following plots for the blue laser e FSC vs SSC dot plot e Histograms for FSC A FITC A FITC H PE A PE Texas Red A PerCP CyS 5 A and PE Cy7 A M Tip Use the sticky buttons feature to easily create multiple plots Press the Control key select a plot tool and click multiple times on the worksheet Each click will create a new plot To deselect the plot tool select another tool or press the Esc key 4 Resize the plots so they fit on one page To display page breaks click on the template background and select the Show Page Breaks checkbox in the Inspector M Tip To resize multiple plots simultaneously resize one plot and then select all the plots you want to change Select the resized plot last so that it has yellow selection handles Click the Resize tool in the Worksheet toolbar mp to make all plots the same size as the selected plot 5 Right click the Blue template in the Browser and choose Create Statistics View Chapter 4 Running Samples 109 6 Edit the Statistics view e Click Edit Statistics View in the Inspector
50. trigons The octagon detects SSC and up to seven fluorescent signals excited by the 488 nm blue laser The trigons detect fluorescent signals excited by the 633 nm red and 407 nm violet lasers respectively Each trigon can detect up to three fluorescent channels An optical upgrade is required to fill all detection channels violet laser signal red laser signal blue laser signal Figure 2 6 Octagon and trigon detector arrays Each detector array houses dichroic and bandpass filters which steer and filter the emitted light and photomultiplier tubes PMTs which detect light signals Chapter 2 Theory of Operation 51 52 Filters Optical filters modify the spectral distribution of light scatter and fluorescence directed to the detectors Two kinds of filters are used in the detector arrays longpass LP filters are used to steer light between the detectors within a detector array while bandpass BP filters in front of each PMT allow fine tuning the spectral wavelengths that need to be collected Figure 2 7 Figure 2 7 Detectors and filters in octagon array BD FACSAria User s Guide Longpass Filters Longpass LP filters pass wavelengths longer than the filter rating and reflect shorter wavelengths For example a 500 LP filter permits wavelengths longer than 500 nm to pass through it and reflects wavelengths shorter than 500 nm Figure 2 8 longpass 480 520 100 4 460 500 540 transmission l T
51. tube holder 345898 Four way 12 x 75 mm collection tube holder 333532 Two way 12 x 75 mm collection tube holder 334904 Two way 15 mL collection tube holder 345896 Spare nozzles e 70 micron set of 3 e 335474 e 100 micron set of 3 e 335475 Nozzle O rings bag of 30 333084 Appendix A Supplies and Options 213 Item Part No Sample injection tubing three 12 in lengths 335598 HPLC valve tubing three 7 in lengths 335599 Ferrules package of 10 335108 Ferrule removal tool 335690 Nozzle lever spring 333081 Nozzle lever plunger 345888 Fluidics cart tubing replacement kit 335457 e 1 4 x 1 8 in sheath tubing 96 in length blue e 1 4 x 1 8 in waste tubing 288 in length orange e 3 8 x 1 4 in air tubing 96 in length clear e 3 8 x 1 4 in waste tubing 96 in length yellow e Fluidics cart power cord Fluid filter assembly Pall SuporCap 50 0 2 um 335663 Bypass tubing assembly tubing and quick disconnect 335760 fittings Adaptor tray for microscope slides 333681 ACDU splash shield 334909 Power cords e Main power cord e 333694 e Cordset for continental Europe e 334140 e Cordset for UK e 334141 e Cordset for Australia Asia e 334175 Allen wrench set 98 10004 00 214 BD FACSAria User s Guide Other Replacement Parts The following items are not included in the accessory kit but you can use the indicated part numbers to order spare or re
52. 0 DN PRIBIO APC 500 OMM ols Add Delete Digital data is displayed on an 18 bit linear scale from 2 6 262 144 You can select the Log checkbox to convert the display to a log scale The Experiment Inspector contains an option to display log data on a four or five decade scale Refer to the BD FACSDiVa Software User s Guide for more information BD FACSAria User s Guide Laser Delay Sample interrogation takes place within the cuvette flow cell As described in Optics System on page 36 fiber optic cables are used to direct laser light through a series of prisms that focus each laser on the stream at a separate position This allows optimal detection of fluorescent signal from each laser with minimal cross contamination from the other beams In a three laser system the red laser intercepts the stream first followed by the blue and then the violet laser Because the laser signals are spatially separated there is a slight delay between the detection of each laser s signal Figure 2 13 cell enters and leaves the first laser intercept red cell enters and leaves the second laser intercept blue cell enters and leaves the third laser intercept i violet time gt Figure 2 13 Signal separation over time The Delay factor in BD FACSDiVa software is used to realign the signals so they can be measured and displayed on the same time scale Note that signals are aligned with respect to the blue
53. 10 2 5 10 10 10 10 CD3 PerCP Cy5 5 A 10 104 10 CD8 APC Cy7 A 10 TB NK_001 Lymphocytes 10 10t 10 CD3 PerCP Cy5 5 A Experiment Name 5 Color Expt Tube Name TIBINK_001 Collection Date CDA FIT CD16 CD3 Pe CD19 A CD8 AP Feb 5 2003 12 18 04 PM Population Events Parent_ Mean Mean Mean Mean Mean E Lymphocytes 2 102 21 0 2 068 825 6 981 700 2 293 MT cells 1 465 69 7 2 936 54 10 005 45 3 155 OT cytotoxic 333 22 7 76 65 8 466 56 13 955 E T Helper 1 074 73 3 3 985 43 10 225 40 21 H nk cells 252 12 0 76 6 155 1 48 647 HB Cells 187 8 9 48 34 19 7 481 48 Figure 4 11 Lymphocyte analysis 134 BD FACSAria User s Guide Reusing the Analysis Now that the analysis strategy has been defined you can use it to analyze the remaining Tubes in the Experiment Acquisition Templates allow you to apply an analysis strategy to a series of data files without saving the analysis each time After previewing the data you can print the analysis or save it to a Tube specific worksheet see the following section Saving the Analysis 1 Move the Acquisition pointer to the next Tube under the LWB Specimen 2 View the data on the Acquisition Template make adjustments to gates as needed NOTICE Adjustments will also apply to the next Tube that is viewed on the template If you don t want to alter the template save the analysis as described in the next section and make adjustments on
54. 33 opening 95 151 assistance technical xi attenuation control 66 automated cell deposition See ACDU backflush sample line 170 bandpass filters 54 BD FACSAria about 21 cytometer 26 fluidics cart 22 workstation 44 beam splitters 53 biohazards xvi bleeding filters 182 breaker circuit 42 228 BD FACSAria User s Guide breakoff drop 61 setting up 141 troubleshooting 141 198 window 62 Browser 78 bulk injection cleaning 176 buttons Acquire 86 chamber light 29 emergency stop 43 Load 86 power 42 See also controls bypass tubing installing 179 C calculating compensation 127 128 cameras about 40 67 cleaning windows 190 cart fluidics 22 chambers ACDU 35 cleaning bulk injection 176 sample injection 29 sort block 32 sort collection 35 changing air filters 187 fluid filters 183 nozzles 140 optical filters 223 sample line 184 charging drops 68 circuit breaker 42 cleaning bulk injection chamber 176 camera windows 190 flow cell 171 fluidics 173 grounding plate 165 instrument 164 168 mode failure 209 modes 168 nozzles 188 optical filters 192 plenum tanks 175 strobe lens 191 clogged nozzle 188 coefficient of variation CV high 207 collection devices 35 91 optics 38 compensation calculating 127 128 gating data 127 Setup 128 Tubes creating 123 components BD FACSAria system 21 BD FACSDiVa workspace 78 87 cytometer 26 electronics 41 fluidics 27 optics 36 workstation 44 computer
55. 71 71 71 E Sort mi Pause Lalo View Counters Tip To pause during sorting click the Pause button Sort counts are retained when you restart sorting by clicking the Pause button again If you need to unload the sample tube during sorting ie to add more sample you can print a Sort Report to keep a record of the current sort counts The sort counts reset to zero when you restart sorting after loading a sample NOTICE When the Sweet Spot is on sorting pauses automatically if the Drop 1 or Gap are out of range This ensures that sorting occurs only under the proper breakoff conditions If a more severe problem such as a clog is detected the stream shuts off and sorting stops the deflection plates shut off the aspirator drawer closes and the sample tube is unloaded To clear a clogged nozzle see Cleaning a Nozzle on page 188 152 BD FACSAria User s Guide Setting Up for Sorting Into a Plate or Slide This section describes how to set up for sorting into a plate or slide For general guidelines see Setting Up for Sorting on page 138 Installing the Sorting Hardware b WARNING Any instrument surface that comes in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling sorting hardware Wear suitable protective clothing and gloves 1 Install the splash shield below the aspirator drawer Close the sort block door and open the sort collection
56. 76 Window Extension 3 000 2 Window Extension 3 000 020203 Blue 020203 Blue Count Count 50 100 150 200 250 50 100 150 200 250 FITC A 1 000 FITC A 1 000 020203 Blue 020203 Blue Count Count 50 100 150 200 250 50 100 160 200 250 FITC H amp 1 000 FITC H amp 1 000 Figure 4 3 Blue laser area scaling before left and after right adjustment 9 Optimize the PMT voltage for each fluorescent parameter Adjust the voltage to achieve a signal of approximately 100 x 10 in the corresponding fluorescence histogram as shown in Figure 4 4 on page 117 114 BD FACSAria User s Guide Recording and Analyzing Data 1 Click Record 2 After all data has been recorded click Unload and remove the tube 3 Draw an Interval gate around each peak on the scatter and fluorescence histograms Vv Tip Ctrl Click the Auto Interval Gate tool and create all Interval gates Then press Escape to deselect the button and adjust the gates as needed 4 Use the Population Hierarchy to rename each population defined by the Interval gates To display the Population Hierarchy view right click the Blue Tube and choose Show Population Hierarchy Select a population in the Hierarchy view and enter a new name to change it For example change P2 to FSC P3 to SSC P4 to FITC and so on Tube Blue Population Call Events BB Singlets X Fsc XK ssc XX Fitc I PE EJ PE TxRd E9 PercP Cy5
57. 97 28 99 117 Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood Body Fluids and Tissue Approved Guideline Wayne PA National Committee for Clinical Laboratory Standards 1997 NCCLS document M29 A Procedures for the Handling and Processing of Blood Specimens Approved Guideline Wayne PA National Committee for Clinical Laboratory Standards 1990 NCCLS document H18 A BD FACSAria User s Guide General Safety A CAUTION Movement of mechanical parts within the instrument can pinch or injure your hands or fingers To prevent injury by moving parts follow these precautions e Keep your hands and clothing away from the sample injection chamber when a tube is loading or unloading Do not place objects underneath the sample injection chamber e Keep your hands away from the sort aspirator drawer during sorting The drawer can close at any time without warning if a clog is detected during sorting When sorting into a plate keep the sort collection chamber door closed during sorting Do not place objects inside the sort collection chamber and do not attempt to access the plate until sorting is complete The microtiter tray stage can move suddenly during the sorting process Sorting continues even when the sort collection chamber door is opened Precaution Labels The following precaution labels appear on the BD FACSAria flow cytometer or fluidics cart to indicate a poten
58. Air lock in filter Recommended Solutions Remove the filter for the corresponding fluid install bypass tubing and run Prime After Tank Refill Repeat the priming procedure until you see fluid in the line When fluid is running through the line remove the bypass tubing install the filter and repeat the priming procedure one last time Long clean fails Air lock in filter See previous recommendations Fluid line detached Verify the fluid line connections on the fluidics cart and on the instrument Push firmly on each line to ensure it is connected Fluidics cart air flow lt 70 psi Air leak Contact your BD Biosciences service engineer Fluidics cart air flow gt 100 psi Regulator not adjusted properly Contact your BD Biosciences service engineer Fluid leak under fluidics cart or below side door Condensation from pressure relief valve This is a normal phenomenon that occurs when water is condensed from room air Condensation is greater in humid environments To avoid slipping check and wipe up the water daily Bleeder valve open Check and close all bleeder valves See Purging Filters on page 182 Broken fluid line Contact BD Biosciences Chapter 7 Troubleshooting 209 Electronics Troubleshooting Observation Possible Causes Instrument Instrument power off Recommended Solutions Turn on the instrument main power Disconnected in Inst
59. HPLC valve primary sample line connecting nut Figure 6 4 Sample line and HPLC valve 184 BD FACSAria User s Guide Use the following steps to replace the tubing The same procedure can be used to replace either the primary or secondary sample line 1 Turn off the stream and lasers if needed make sure the sample injection chamber is in the unload position 2 Unscrew the connecting nuts on each end of the tubing and pull out the sample line 3 Ensure that a cone shaped ferrule is attached to each end of the sample line Figure 6 5 on page 186 shows an example of a ferrule If the ferrule was left behind in the injection chamber fitting the HPLC valve or the flow cell fitting screw the tip of the ferrule removal tool included in the accessory kit into the top of the ferrule and pull the ferrule straight out After using the tool you will need to replace the ferrule spares are included in the accessory kit ferrule removal tool 4 Slide the ferrule and nut off each end of the sample line Vv Tip Handle the ferrule with care it is small and easily lost 5 Slide the nut and then the ferrule onto each end of the new sample tubing e If you are replacing the primary sample line leave approximately 0 1 in 0 25 cm of tubing extending out of the ferrule on the HPLC end and approximately 5 in 12 7 cm of tubing extending out of the sample injection chamber end This length can be adjusted depending on the depth o
60. Layout eremita or iaa ia 90 Sort Report uscar rosas pasas as 97 Chapter 4 Running Samples 99 Starting Up the Instrument 0 0 cece eect o 100 Setting Up the Fluidics Cart 0 0 cece cece eee eens 102 Instrument Quality Control 1 0 0 ccc ee ee eee A 105 Setting Up the Experiment 00 cece eee eee eee momo 105 Verifying Area Scaling and Laser Delay o ooooooooomom oo 112 Reusing the Experiment 0 0 c cece eee eee teen ences 120 Sample Optimization Using Instrument Setup 0 00202 eee 121 Creating the Experiment 0 c cece e nneur ernn 122 Adjusting the Voltages and Threshold oooooooooooo o o 124 Calculating Compensation 0 0 e cece eee eee eens 127 Recording and Analyzing Data 0 ccc cc ee teenies 129 Setting Up the Acquisition Template 0 cece cece eens 129 Recording Data 0 ccc cc ence ent een E AE 131 Analyzing Data iia dia 132 Reusing the Analysis 1 0 0 ccc cece cece e nee eens 135 Saving the Analysis 0 0 ccc ccc cece E EERE REEE 135 Chapter 5 Sorting 137 Setting Up for Sorting 0 0 ec e eee erer 138 Changing the Nozzle 1 0 0 0 c ccc cence nee e eens 140 Setting Up the Breakoff 0 ccc ccc ee eee eens 141 Setting Up for Bulk Sorting 2 0 0 ee eee eens 143 Determining the Drop Delay 0 cece eee eee eens 145 Setting Up the Experiment 0 0 cece cent eee ees 145
61. PS Detector Fluorochrome LP Mirror BP Filter Blue H Blue G Blue F Blue E Blue D Blue C Blue B Blue A Red C Red B Red A Violet C Violet B Violet A 222 BD FACSAria User s Guide Changing Filters Note that two extra filters are provided in the accessory kit e If you are using PerCP rather than PerCP CyS 5 in the Blue B position replace the 695 40 filter with the 675 20 filter to decrease the amount of spectral overlap e If you are using both PE and PE Texas Red in your experiment replace the 585 42 filter in the Blue D position with the 575 26 filter to optimize the detection of each fluorophore If you want to install a custom filter or dichroic the filter should comply with the following specifications Table A 1 BD FACSAria filter specifications Filter Characteristic Dichroic LP Filters BP Filters Diameter 0 622 0 003 in 1 00 0 010 in Thickness 0 125 0 005 in 0 12 0 35 in Minimum clear aperture 0 562 in 0 85 in Incident angle 11 1 0 For the longpass filters the surface that faces into the center of the octagon trigon should be coated directly on its surface not between two or more pieces of glass The coating should transmit gt 70 of the wavelength range you want the filter to transmit with a minimum transmission of gt 50 and it should reflect gt 90 of the wavelength range you want the filter to ref
62. Running Samples 9 3 9 5 2 7 2 7 2 7 2 7 2 7 97 474 87 97 508 8 9 96 748 2 6 96 745 2 5 96 749 2 5 96 756 2 5 96 749 25 117 Optimizing Signal from the Red Laser 1 Double click the Red template icon and move the Acquisition pointer to the Red Tube in the Browser Install the tube containing fluorescent particles for the red laser onto the loading port click Load in the Acquisition Controls frame Set the Flow Rate to 1 0 Adjust the FSC gain and the SSC voltage to place the particles on scale in the FSC vs SSC dot plot Draw a region around the singlet population Select all histograms on the Red template and select the P1 checkbox in the Inspector The histograms change to display data from only the Singlet population Adjust the APC voltage to place the APC H signal at approximately 100 x 10 Optimize the Delay setting for the red laser Laser Delay settings vary depending on the sheath pressure the delay time is shorter at higher sheath pressures Adjust the delay to synchronize laser signals in time The delay has been properly adjusted when the fluorescent signal intensity is at its highest e Draw an Interval gate around each peak on the fluorescence histograms e Select the Laser tab in the Instrument frame Change the Window Extension to zero to capture pulses within the narrowest time window e Adjust the red laser Delay setting in increments of 0 1 to obtain the highest mean channel
63. Stream window The Side Stream window displays an image of the side streams as transmitted by the lower camera In addition to the stream image the Side Stream window contains the controls used to adjust electrical charges and to determine the drop delay using Accudrop as described in Setting the Drop Delay on page 67 High Voltage Bl Test Sort al Optical Filter E Attenuation Drop Delay 43 76 tl rr Y Voltage Center E Plate Voltage 5 000 2nd Drop 201 3rd Drop 1041 4th Drop 53t Phase 031 Figure 2 16 Side Stream window Controls in the Side Stream window are described in Table 2 2 on page 66 Preprogrammed values can be downloaded to the Side Stream window by choosing High Medium Low or Custom from the Sort gt Sort Setup menu Note that changes to values in the Sort Setup windows Side Stream and Breakoff are automatically saved At startup the last settings used on the instrument are restored except the states of the Voltage Test Sort and Optical Filter buttons which always default to off Default values can be restored by choosing Sort gt Sort Setup gt Restore Defaults For more information see Sort Setup on page 89 Mi Tip To save screen space you can hide the controls in the lower half of the window To do so right click the window and choose Hide Controls from the contextual menu You can also choose to hide button labels To show the controls again right click the window and
64. T T 450 500 550 wavelength nm E Figure 2 8 Light reflection by longpass filter Dichroic filters that are used to direct different color light signals to different detectors are called dichroic mirrors or beam splitters Although dichroic mirrors have the properties of LP optical filters you cannot necessarily use any type of LP filter as a beam splitter A beam splitter must have a surface coating that reflects certain wavelengths but many types of LP filters are absorbance filters that do not have any specific reflective characteristics Also optical filters and beam splitters are rated at a specific angle of incidence When used as a beam splitter they are placed at an angle relative to the light source Their optical properties are therefore designed for that angle of incidence The detector arrays use LP dichroic mirrors to steer progressively shorter wavelengths of light to the next PMT in the array For example in the octagon array light first passes through a 735 LP filter in the A position followed by a 655 LP filter in the B position Thus wavelengths longer than 735 nm are detected at PMT A all wavelengths lt 735 nm are reflected to PMT B Wavelengths between 655 nm and 735 nm are detected at PMT B all wavelengths lt 655 nm are reflected to PMT C and so on For a list of the longpass filters used in the detector arrays see Table 1 1 on page 40 Chapter 2 Theory of Operation 53 54 Bandpass Filters
65. User s Guide Acquisition Troubleshooting continued Observation Distorted parameters or high CVs Possible Causes Instrument settings adjusted incorrectly Recommended Solutions Optimize the scatter parameters See Adjusting the Voltages and Threshold on page 124 Flow rate too high Decrease the Flow Rate in the Acquisition Controls frame Window Extension too low Increase the Window Extension Bubbles in flow cell Turn off the stream wait a few seconds and turn on the stream again Nozzle clogged or dirty Clean the nozzle as described in Cleaning a Nozzle on page 188 Flow cell dirty Clean the flow cell with a detergent such as Contrad See Clean Flowcell on page 171 Let the detergent sit for 5 minutes before turning on the stream Poor sample preparation Repeat sample preparation Area scaling factor too low Verify area scaling See Verifying Area Scaling and Laser Delay on page 112 Excessive amount of debris in plots Threshold channel too low Increase the Threshold channel See Adjusting the Voltages and Threshold on page 124 Dead cells or debris in sample Examine the sample under a microscope Sample contaminated Re stain the sample making sure the tube is clean Chapter 7 Troubleshooting 207 Acquisition Troubleshooting continued Observation Possible Causes High electronic abort rate Window Extension too high
66. a higher flow rate is generally used for qualitative measurements such as immunophenotyping the data is less resolved but is acquired more quickly A lower flow rate is generally used in applications where greater resolution is critical such as DNA analysis For sorting applications sorting results are optimized at lower flow rates lt 8 0 Signal Generation The following sections describe how signals are generated when cells or particles intercept the laser within the cuvette flow cell Light Scatter When a cell or particle passes through a focused laser beam laser light is scattered in all directions Light that scatters axial to the laser beam is called forward scatter FSC light that scatters perpendicular to the laser beam is called side scatter SSC FSC and SSC are related to certain physical properties of cells e FSC indicates relative differences in the size of the cells or particles e SSC indicates relative differences in the internal complexity or granularity of the cells or particles side scatter forward scatter light source Chapter 2 Theory of Operation 49 Fluorescent Signal When cells or particles stained with fluorochrome conjugated antibodies or other dyes pass through a laser beam the dyes can absorb photons energy and be promoted to an excited electronic state In returning to their ground state the dyes release energy most of which is emitted as light This light emission is know
67. about 44 shutting down 164 starting 100 configuration instrument adding 224 three laser 221 two laser 220 conflicts sort about 69 counting 95 printing 97 saving 93 troubleshooting 202 connecting external air 24 fluidics cart 23 power cords 23 42 containers about 23 refilling 103 containing aerosols 34 controls ACDU stage 95 acquisition 79 86 aspirator drawer 95 attenuation 66 compensation 123 fluidics 80 hiding 64 65 instrument 79 optical filter 66 67 sorting 87 94 stream 63 Sweet Spot 63 test sort 66 voltage 66 See also buttons conventions user s guide x cooling samples 82 102 copying Analysis objects 135 cord damaged xv fluidics cart 23 25 counters sort 95 Index 229 creating Acquisition Templates 108 129 Analysis objects 132 compensation Tubes 123 custom devices 88 158 Experiments 106 folders 106 gates 115 plots 109 Sort Layouts 92 149 Sort Precision modes 75 statistics view 109 custom devices 88 158 cuvette flow cell about 30 cleaning 171 cytometer about 26 cleaning 164 168 disconnect error 210 doors 26 electronics 41 fluidics 27 46 optics 36 power 42 shutting down 162 starting 100 D damaged cord xv data analyzing 115 129 132 gating 115 127 132 recording 115 129 131 deflection drop 69 plates 33 troubleshooting 200 230 BD FACSAria User s Guide delay adjusting 118 drop 66 67 145 See also Accudrop laser 59 deleting custom
68. aining commands to move the tray support arm either manually or to the home position see Setting Up the Stream on page 155 Custom Devices opens a dialog box where custom devices can be defined see Creating a Custom Device on page 158 Sort Report displays a report showing the sort settings acquisition counters and Sort Layout information from the current sort See Sort Report on page 97 BD FACSAria User s Guide Sort Setup Sort setup values for high medium and low pressure sorting can be downloaded using the Sort gt Sort Setup command Default settings are provided for each sorting option along with Custom settings for non sorting applications Use the following guidelines to determine which Sort Setup option to use Sort Setup Nozzle um Recommended Applications High 70 High speed high throughput sorting Medium 70 Sorting of cell lines fragile cells Low 100 Single cell sorting ACDU sorting sorting of large or fragile cells Custom 70 Non sorting mode for high sensitivity If you make changes to any of the settings changes are automatically saved when you quit BD FACSDiVa software or when you switch to a different Sort Setup mode When you restart the most recently used set of values is restored You can restore default High Medium or Low settings by choosing Sort gt Sort Setup gt Restore Defaults Default settings are shown in Table 3 1 Table 3 1 Default Sort Setup values
69. al decontaminant after performing a Long Clean with bleach or ethanol This will ensure a safe environment for biological samples 8 When startup is complete choose Instrument gt Fluidics Shutdown 9 When shutdown is complete choose Instrument gt Fluidics Startup again 10 Optional When the startup is complete turn on the stream and let it run for 20 30 minutes Cleaning the Plenum Reservoirs After you run a Long Clean with bleach or ethanol you can clean the plenum reservoirs by autoclaving them or by wiping them down with ethanol or DI water Use the following procedure to remove and clean the reservoirs 1 Turn off the instrument main power 2 Open the side door and verify that the plenum reservoirs are empty 3 Place absorbent paper under the plenum reservoirs and remove each reservoir by unscrewing it from its receiver plenum reservoir Chapter 6 Shutdown and Maintenance 175 4 Pull off the tubing connected to each reservoir tank 5 Autoclave the reservoirs or wipe them down with 70 ethanol followed by DI water After wiping them down dry them with a clean lint free cloth 6 Clean the float and reservoir receiver for each plenum tank Use a spray bottle to clean the floats use a cloth soaked with ethanol to wipe down the receivers A NOTICE The floats are fragile Handle them with care Do not spray ethanol into the instrument behind or below the plenum reservoirs Dry each float and reservoir with a
70. am voltage Recommended Solutions Increase the voltage for the far left stream Splash shield not installed Install the splash shield See Installing the Sorting Hardware on page 153 No deflection or Insufficient voltage insufficient deflection e Increase the plate voltage e Increase the side stream voltages using the slider controls Stream charging wire loose or missing Verify the stream charging wire is inserted all the way into the nozzle Salt bridge Turn off the stream Remove the nozzle and the grounding plate and thoroughly clean and dry the plate and the area around the plate See Cleaning the Nozzle and Grounding Plate on page 165 for instructions Plates too far apart at top Adjust the plates so there is a gap of approximately 0 28 in 0 7 cm between them at the top Sorting paused because actual Drop 1 value out of range Wait until the Sweet Spot adjusts the amplitude to achieve the Drop 1 target Tf this happens repeatedly during sorting you could have debris in the nozzle or flow cell See Troubleshooting the Stream on page 194 for suggestions 200 BD FACSAria User s Guide Sorting Troubleshooting continued Observation Fanning around center or Possible Causes Nozzle inserted improperly Recommended Solutions Turn off the stream Remove the side streams nozzle and ensure that the O ring is in place Reinsert the nozzle at a slight downw
71. amplitude value results in a shorter stream breakoff a lower amplitude results in a longer stream breakoff Typically the amplitude is set once at the beginning of a sorting experiment and then maintained via the Sweet Spot Frequency field the size of the drops Drop size is also influenced by the nozzle size The drop drive frequency can be adjusted from 1 0 102 0 kHz The higher the frequency the more drops are generated per second and the smaller the drops The lower the frequency the fewer drops generated per second and the larger the drops Determines the number of drops formed per second and O cap 6 7 Chapter 2 Theory of Operation 63 64 Table 2 1 Breakoff window controls continued Control Description Frequency field In general the drop drive frequency should not need adjustment continued BD Biosciences recommends using the default values that are input with each Sort Setup mode Drop 1 field The distance between the top of the image and the center of the first broken off drop from 100 600 pixels When you input a value and turn on the Sweet Spot the instrument automatically adjusts the amplitude to attain your target value Note that not all Drop 1 targets are attainable The Drop 1 value jumps in whole increments of approximately 57 pixels according to your drop spacing The same Drop 1 setting should be used from day to day a difference of up to 10 pixels between t
72. an external source although the cart can be hooked up to an in house air source if one is available The air pumps provide pressure from 2 to 75 psi to accommodate a variety of cell sorting applications Air pressure is adjusted within BD FACSDiVa software 10 L container 5 L container pressure gauge door access knob 7 filter Figure 1 2 Fluidics cart 22 BD FACSAria User s Guide Containers and Connectors The fluidics cart holds four 10 L containers two sheath and two waste and three 5 L auxiliary cleaning fluid containers Figure 1 3 All containers caps level sensors and tubing are autoclavable The cart can also accommodate up to two BD FACSFlow 20 L cubitainers in place of the four 10 L containers sheath port waste tank sheath tank Figure 1 3 Fluidics cart containers NOTICE To prevent foaming do not fill the containers with detergent containing solutions The fluidics cart connects directly to the flow cytometer unit via a power cord fluid hoses and air line Figure 1 4 on page 24 Receptacles for the aerosol management and temperature control options are also located within the connection panel The position of the fluidics cart is constrained only by the length of the power cord and fluidics hoses which extend up to 8 feet 2 4 m Typically the cart is placed to the left or underneath the cytometer Chapter 1 BD FACSAria Instrument Components 23 fluidics cart serial communication
73. ard angle to prevent loss or movement of the O ring Make sure the nozzle is completely seated against the back wall of the cuvette flow cell Sweet Spot off Turn on the Sweet Spot Incorrect Sort Precision mode Verify that the Sort Precision mode is appropriate for your sorting requirements See Sort Precision Modes on page 73 2nd 3rd 4th Drop values not optimized Adjust the 2nd 3rd and 4th Drop settings to tighten the center stream and fine tune the side streams Particles too big for nozzle Change the nozzle See Changing the Nozzle on page 140 Sort button disabled Acquisition pointer not set to current Tube Click to move the Acquisition pointer in front of the appropriate Tube Population not listed in Add menu on Sort Layout Population defined using Snap To gate Redefine the population using another gate type Viewing Sort Layout for another Tube Open or create a Sort Layout for the current acquisition Tube Sort Layout counters not updating Viewing Sort Layout for another Tube Open or create a Sort Layout for the current acquisition Tube Chapter 7 Troubleshooting 201 Sorting Troubleshooting continued Observation High sort conflict rate Possible Causes Event rate too high for drop drive frequency Recommended Solutions Decrease the event rate Gating conflict Verify the gating hierarchy Purity Mask too high Decr
74. arges drops how the drops are deflected and how sorting populations are identified BD FACSAria User s Guide Sample generates light scatter and fluorescent signal signal is analyzed Charge is applied via the stream charging wire in the nozzle The charged droplet breaks off Deflection plates attract or repel charged droplet Uncharged droplets pass to waste Charged drops containing particles of interest are collected 000000 Figure 2 15 Sorting Drop Formation The BD FACSAria instrument is unique in that drop drive energy is constantly applied to the stream droplets form as soon as you turn on the stream Sample interrogation takes place upstream of the stream vibration so that analysis is not affected by the drop drive A drop breakoff image is created using an LED strobe and a video camera The image is displayed in the Breakoff window See Breakoff Window on page 62 Patented Sweet Spot technology analyzes the drop breakoff image and provides feedback to the appropriate instrument controls US Patent No 5 700 692 Chapter 2 Theory of Operation 61 62 Breakoff Window The upper camera transmits an image of the drop breakoff to the Breakoff window where video image processing software converts the visual characteristics of the image into numerical properties The drop breakoff is analyzed for two key features
75. ater than zero the Yield Mask automatically reverts to zero 72 BD FACSAria User s Guide Sort Precision Modes Mask values can be combined in many different ways By default five Sort Precision modes are already defined Purity Yield Single Cell Initial and Fine Tune Precision Mode Puy y Yield Mask 32 Purity Mask E Phase Mask Lona Single Cell E Add Delete Close Precision Mode Single Fine Purity Yield Cell Initial Tune Yield Mask 32 32 0 32 0 Purity Mask 32 0 32 0 0 Phase Mask 0 0 16 0 0 Single Cell O O O O e In Purity mode the Yield Mask is set to the maximum to obtain the greatest number of particles because the Purity Mask is also at the maximum only drops with a target particle will be sorted Sorting in Purity mode results in a sorted sample that is highly pure at the expense of recovery and yield e In Yield mode only the Yield Mask is used at its maximum value thus recovery and yield are optimized at the expense of purity e In Single Cell mode the Purity Mask is set to the maximum so only drops containing a target particle will be sorted The Phase mask is set at half the maximum so only particles centered within the sorted drop are deflected Drop trajectory and count accuracy are optimized at the expense of yield Chapter 2 Theory of Operation 73 74 This mode is recommended for single cell sorting or situations where precise cou
76. ator 33 emptying 104 width parameters 58 window extension 118 windows breakoff 62 side stream 65 See also frames views worksheet frame 79 workspace components 78 87 toolbar 78 workstation about 44 shutting down 164 starting 100 Y Yield Masks about 69 Phase Masks using with 70 72 Yield mode 73 Index 237 238 BD FACSAria User s Guide
77. ce 173 Choose from the following Long Clean Select a cleaning method C Long Clean with Bleach Long Clean with Ethanol C Clean Bulk Injection Chamber Cancel NOTICE For the bulk injection chamber option see Clean Bulk Injection Chamber on page 176 5 Wait 8 10 minutes for the cleaning cycle to finish click OK to dismiss the completion message Y Long Clean Status G Long clean is complete Before turning on the stream run Fluidics Startup at least once to remove trace decontaminant from the sheath path Refer to the user s guide for complete instructions NOTICE If the completion message does not appear after 10 minutes verify that there are no error messages in the Status tab of the Instrument frame If the cleaning mode fails see Fluidics Cart Troubleshooting on page 209 6 Optional Remove the plenum reservoirs and wipe them down with bleach or ethanol followed by DI water BD Biosciences recommends that you remove the plenum reservoirs only for the most stringent decontamination You do not need to perform this step each time you run a Long Clean If needed see Cleaning the Plenum Reservoirs on page 175 174 BD FACSAria User s Guide 7 Choose Instrument gt Fluidics Startup The Fluidics Startup procedure removes cleaning fluid from the plenum tanks and fluid lines and replaces it with sheath fluid The following steps are not required but are recommended for removing residu
78. charge from the first drop Single Cell D degrades the quality of the side streams Thus Add Delete Close when four way sorting or sorting into small wells where precise deflection is required a Yield Mask of zero is recommended For more information see Yield Mask on page 69 1 In the Browser right click the Tube containing the defined population subset s to be sorted and choose New Sort Layout Alternatively select a Tube in the Browser and click the Sort Layout button in the Workspace toolbar Ef By default the 2 Tube Sort Layout appears Chapter 5 Sorting 149 2 Make appropriate entries in the Sort Layout view 4 Tube Sort Layout_001 Eg Device Precision Target Events Save Conflicts b Tube bd Purity y y LJ Sort Rate NA Confl Cnt NA Confl Rate NA Efficiency NA BN Sort mm Pause az ja View Counters e Choose the collection device from the Device menu e Change the Sort Precision mode to Purity two tubes Four Way Purity four tubes or Single Cell plate or slide e Enter the number of Target Events by choosing a value from the drop down menu or entering a number in the field e Select the sort location field s to be sorted into Select multiple fields by dragging the mouse select a row or column by clicking the row or column header e Add the required population s to each sort location field If the Add menu doesn t appear after select
79. choose Show Controls Chapter 2 Theory of Operation 65 66 Table 2 2 Side Stream window controls Control Voltage button Test Sort button Al on off Optical Filter button g q out Attenuation button E aa on off Drop Delay field Voltage sliders far left left right far right Voltage Center slider Plate Voltage field 2nd 3rd 4th Drop fields Phase field Description Turns the plate voltage on and off O by clicking Generates test side streams based on test sort pulses when the button is clicked Controls the position of the optical filter in front of the lower Accudrop camera Decreases the amplitude of the drop drive when the button is clicked At startup attenuation defaults to off As a general rule turn on attenuation when sorting below 30 psi Sets the amount of time between when an event is measured and the breakoff point from 10 140 drops The drop delay value determines which drop will be deflected The drop delay value is set experimentally using BD FACS Accudrop technology Set the percentage of charge to be applied to the corresponding stream as a percentage of maximum Adjusts the relative voltage between the left and right plates which moves the streams slightly to the left or right as a whole Adjusts the total voltage difference between the plates which determines the angle of stream deflection Apply a correction factor for the drop charge as a percentage
80. clean lint free cloth 7 Reinstall the plenum reservoirs Screw each reservoir to its receiver while making sure that the O rings are seated properly within each plenum tank Reattach the tubing to the bottom of each reservoir Clean Bulk Injection Chamber Use the Clean Bulk Injection Chamber option to flush the injection chamber with DI water and ethanol NOTICE Allow 5 10 minutes to complete the cleaning procedure 1 Ensure fluid levels are adequate empty the waste container if it is full This procedure requires approximately 250 mL each of DI water and ethanol 2 Turn off the lasers and the stream if they are on 3 Remove the tube holder from the loading port It can be cleaned separately by wiping it down with ethanol if needed 176 BD FACSAria User s Guide 4 Choose Instrument gt Cleaning Modes gt Long Clean 6 If you have not already turned off the stream a message appears reminding you to do so Select the Clean Bulk Injection Chamber option click OK Long Clean Selecta cleaning method Long Clean with Bleach Long Clean with Ethanol Clean Bulk Injection Chamber Cancel During the next 4 5 minutes the instrument performs the following e closes and seals the bulk injection chamber e fills the chamber with DI water and flushes the chamber e fills the chamber with ethanol and flushes the chamber o air dries the chamber and then opens it A message appears when cleaning is c
81. cs Cart on page 102 If you disconnect the DI water tank for refilling prime the system before continuing 162 BD FACSAria User s Guide 7 8 The instrument starts the Daily Clean cycle Daily Clean removes sheath from the lines and plenum and replaces it with DI water When prompted install a tube containing approximately 3 mL of cleaning fluid such as BD FACSRinse solution BD Catalog No 340346 and click OK confirm Q Install a tube with BD FACSRinse solution on the loading port and click OK Cancel The instrument loads the tube and cleans the cuvette with the cleaning solution A progress message appears during the cleaning process click OK to dismiss the message if you would like t i Shutdown Status Please wait When prompted install a tube containing approximately 3 mL of DI water click OK confirm 2 Install a tube with DI water on the loading port and click OK Cancel The instrument loads the tube and rinses the cuvette with DI water A progress message appears during the cleaning process click OK to dismiss the message if you would like Chapter 6 Shutdown and Maintenance 163 9 Click OK when you see a message informing you that the system can be turned off Shutdown Status G Itis now safe to turn off your system Turn off the instrument main power quit BD FACSDiVa software and shut 10 down the computer External Cleaning To
82. ctly Verify that the correct Boolean logic And Or was used for the Threshold parameters Threshold channel too low or too high Adjust the Threshold channel See Adjusting the Voltages and Threshold on page 124 Optical filter s not completely seated Make sure the filters are pushed all the way in 204 BD FACSAria User s Guide Acquisition Troubleshooting continued Observation No fluorescent signal Possible Causes Current Instrument Configuration different from optical setup Recommended Solutions Verify that the instrument optics setup matches the current Instrument Configuration Wrong filter installed or filter not completely seated Make sure the appropriate filter is installed for each fluorochrome see Application Options on page 220 for suggestions Make sure the filters are pushed all the way in Laser delay set incorrectly Adjust the laser delay settings See Instrument Quality Control on page 105 Low Area signal Area Scaling factor too low Adjust Area Scaling for the corresponding laser See Instrument Quality Control on page 105 Unexpected events in plot Incorrect logic in Population Hierarchy Verify the gating strategy Incorrect population s in plot Right click the plot and choose Show Populations Verify that the appropriate populations are displayed Incorrect drawing order Verify that the required population is not hidde
83. cts Continuous For a four tube layout conflicts for the Far Left tube are sorted to the left conflicts for the Far Right tube are sorted to the right No other populations can be sorted into the center most tubes Device Precision Target Events Save Conflicts 4 Tube gt Purity HH y Far Left Right Far Right Left Conflicts Contin Right Conflicts Cont Editing a Sort Layout e To change the number of events for any population click on the Sort Location field s containing the population and then choose a number from the Target Events field or enter a new number e To remove a population from a sort location field select the field and then choose the corresponding population from the Delete menu e To clear all populations from a field select the field and then choose Clear All Using Sorting Controls Sorting controls appear at the bottom of the Sort Layout window Use these controls for the following functions oms ge Pace aj e Sort starts sorting events for the current acquisition Tube All counters reset to zero when this button is clicked Events are sorted until the requested number of sorted events has been reached 94 BD FACSAria User s Guide Click the Sort button again to stop sorting before reaching the requested number of events the counters stop at the number of sorted events If you click Sort to restart sorting the counters reset to zero e Pause stops sorting but
84. d by laser and fluidics performance BD strongly recommends following the laser and fluidics maintenance procedures in Chapter 6 Setting Up the Experiment The steps in this section show you how to set up an Experiment for instrument QC If you have already created a similar Experiment you can reuse it by duplicating the most recent Specimen See Reusing the Experiment on page 120 1 Choose Instrument gt Instrument Configuration and verify the current configuration Make sure the configuration lists appropriate parameters and that the lasers and detectors reflect the setup of the detector arrays A NOTICE For accurate data results the instrument optics setup must match the current Instrument Configuration Chapter 4 Running Samples 105 2 Deselect all options in General User Preferences N User Preferences General Gates TF Tube specific worksheet I Start Acquisition on pointer change I Remove Tube Specific Instrument Settings on Duplicate Acquisition Template I Default Acquisition Template I Save Analysis After Recording EN 3 Click the corresponding tools in the Workspace toolbar to display the Browser Instrument Inspector Worksheet and Acquisition Controls frames as needed galaa 4 Optional Create a folder for your Experiment Select the top level Experiments icon in the Browser press Ctrl N to add a new folder top level icon 33 Experiments folder E i Instrument QC Renam
85. data is recorded 136 BD FACSAria User s Guide 5 Sorting You can program BD FACSDiVa software to sort a specified number of particles from multiple populations into a variety of sorting devices including tubes plates and slides Collection devices are provided for sorting into 1 mL 12 x 75 mm and 15 mL tubes hardware for sorting into plates and slides is available as an option Up to four defined populations can be sorted into each tube allowing up to 16 populations to be sorted at one time Any subsetted population can be used for sorting including populations defined by Quadrant gates Interval gates or derived gates A single sort population can be defined by up to eight gates The following topics are covered in this chapter e Setting Up for Sorting on page 138 e Determining the Drop Delay on page 145 e Sorting on page 149 e Setting Up for Sorting Into a Plate or Slide on page 153 Before you begin you should be familiar with BD FACSAria operation using BD FACSDiVa software Review chapters 2 and 3 if needed 137 Setting Up for Sorting In general do the following to set up for a sorting experiment Each step is explained in more detail in previous or subsequent sections 1 Install the appropriate size nozzle As a general guideline the nozzle opening should be at least three times the diameter of the particle to be sorted See Changing the Nozzle on page 140 Start up the instrument the computer
86. deflection plate voltage turn off the lasers and stream 2 Open the sort block door and remove the grounding plate under the nozzle Mi Tip Use the wooden end of a cotton swab to pull the grounding plate toward you 3 Pull the stream charging wire out of the nozzle See Figure 6 1 on page 166 Chapter 6 Shutdown and Maintenance 165 stream charging wire Figure 6 1 Removing the stream charging wire 4 Turn the nozzle locking lever to the left and pull the nozzle out of the cuvette flow cell Mi Tip To avoid losing the O ring hold the nozzle steady and pull it straight out without tilting it from side to side If you do not see the O ring on the nozzle it could be stuck to the cuvette Use the wooden end of a cotton swab to locate and remove the O ring 5 Turn the nozzle locking lever 180 to the right while holding your left index finger along the left side of the lever pull out the lever NOTICE There is a compressed spring at the end of the lever Keep your finger alongside the lever to prevent the spring from shooting out when it is released If you lose the spring or plunger spare parts are included in the accessory kit 6 Remove the spring and plunger from the end of the lever Figure 6 2 hole eae spring plunger lever Figure 6 2 Disassembling the lever 166 BD FACSAria User s Guide 7 10 Clean the disassembled parts and the exposed instrument surfaces with DI water Clean
87. devices 160 sort populations 94 Sort Precision modes 75 detectors 39 51 devices sorting 91 diode laser 41 67 discriminating filters 54 doors flow cell access 26 instrument 26 sort collection chamber 35 drop breakoff 61 charging 68 conflicts 69 correction factors 66 144 deflection 69 delay 66 67 145 See also Accudrop satellites 142 Drop 1 about 62 64 adjusting 64 142 E editing Sort Layouts 94 statistics view 110 electrical safety xv electronics aborts 208 about 41 troubleshooting 210 emergency button 43 emission spectra 50 emptying waste 104 error messages cleaning mode failure 209 instrument disconnected 210 instrument not responding 210 Master DAQ overflow 210 events not showing in plots 203 205 rate troubleshooting 206 troubleshooting 208 excitation optics 36 Experiment Layout 108 130 Experiments Accudrop 145 creating 106 immunophenotyping 129 instrument QC 105 reusing 120 sample optimization 122 exporting Sort Reports 98 F FACSAria See BD FACSAria ferrules removing 185 fiber optics 37 filters about 52 air changing 187 bandpass 54 changing 223 default setup 40 220 221 discriminating 54 fluid changing 183 longpass 53 optical cleaning 192 purging 182 removing 183 Fine Tune mode 74 flow cell about 30 access door 26 cleaning 171 flow rate adjusting 48 86 recommendations 49 fluidics about 46 cart 22 cleaning 173 components 27 connections 23 containers 23 co
88. diant energy induced damage to the outer epidermal cell layer of the cornea These effects can be the result of laser exposure lasting only a fraction of a second xiii Laser Classification Laser hazard levels depend on laser energy content and the wavelengths used A numbered system is used to categorize lasers according to different hazard levels The higher the classification number the greater the potential hazard The BD FACSAria flow cytometer is a Class I laser product per 21 CFR Subchapter J and EN 60825 1 1994 A2 2001 The lasers are fully contained within the instrument structure and call for no special work area safety requirements except during service procedures These procedures are to be carried out only by BD Biosciences service personnel Precautions for Safe Operation A WARNING Modification or removal of the optics covers or laser shielding could result in exposure to hazardous laser radiation To prevent irreparable damage to human skin and eyes do not remove the optics covers or laser shielding adjust controls or attempt to service the instrument any place where laser warning labels are attached see Precaution Labels on page 1 A CAUTION Controls adjustments or performance of procedures other than those specified in the BD FACSAria User s Guide can result in exposure to hazardous visible or ultraviolet laser radiation or both A caution Keep all instrument doors closed during instrument operation When op
89. dicated by page 171 the flow cell with a distorted scatter high CVs decontaminant Prime After Tank Refillon Primes the fluid lines for the When a fluidics line is page 172 designated fluid s unplugged to refill a tank Long Clean on page 173 Decontaminates the internal Monthly sheath path with bleach or ethanol and rinses it with DI water Clean Bulk Injection Decontaminates the sample Monthly Chamber on page 176 injection chamber with ethanol Prepare for Aseptic Sort Decontaminates the complete When needed before on page 178 sheath path with ethanol and aseptic sorting rinses it with DI water A NOTICE Some of the internal cleaning modes require that you turn off the stream Remember to always turn off the lasers before you turn off the stream Running the lasers without the stream can degrade the performance of the cuvette flow cell Chapter 6 Shutdown and Maintenance 169 Sample Line Backflush After a sample tube is unloaded the sample line tubing within the sample injection chamber is automatically flushed inside and out with sheath fluid to eliminate potential sample carryover Use the Sample Line Backflush command to perform additional backflushing of the inside of the sample line after a tube unloads Perform the Sample Line Backflush when you observe sample carryover or after running samples with adherent cells or dye NOTICE Keep the stream running while performing the backflush 1 Choose Instrument gt Cleaning Mode
90. disabled remove the filter install bypass tubing and repeat the priming procedure until you see fluid in the line When fluid is running through the line remove the bypass tubing install the filter and repeat the priming procedure one last time Communication failure between workstation and instrument Quit the software and restart it Chapter 7 Troubleshooting 195 Troubleshooting the Stream continued Observation Possible Causes No stream when Stream Sheath container low or empty control clicked Recommended Solutions Refill the sheath tank See Refilling Containers on page 103 Note that when the empty tank warning message is not dismissed after 15 minutes the stream shuts off automatically Air in sheath line Prime the sheath tank See Prime After Tank Refill on page 172 Air in filter Purge the filter See Purging Filters on page 182 Dry filter 1 Install bypass tubing in place of the filter for the affected fluid and run Prime After Tank Refill 2 Reinstall the filter and open the bleeder valve to purge the filter See Purging Filters on page 182 Fanning around center Nozzle inserted improperly stream Reinsert the nozzle Push it gently all the way forward without rocking it from side to side Unstable stream Debris in flow cell or nozzle Remove the nozzle and run the stream with no nozzle in place for approximately 1 second Click the Stream con
91. e BD FACSDiVa Software User s Guide the following acquisition controls are available for the BD FACSAria instrument HE Acquisition Controls x Blue 696 evtis 00 02 26 Next Bl Load Acquire Record Restart Storage Gate C All Events v Stopping Gate lo All Events v Events To Record 10000 evt bg Events To Display 500 evt T Flow Rate 1 0 El e Load lifts a tube into the sample injection chamber starts sample agitation if agitation is turned on and starts acquisition of the sample When a tube is already loaded the button changes to Unload Clicking Unload stops acquisition of the sample turns off the agitator and lowers the tube from the sample injection chamber NOTICE The Load button is enabled only when the workstation is connected to the instrument an Experiment is open and the stream is turned on e Acquire stops sample acquisition without unloading the sample tube The Acquire button is disabled when there is no tube loaded e Flow Rate controls the sample flow rate from 1 0 11 0 Do one of the following to change the flow rate Select the value in the field and enter a new value Click the pointer in the slider bar and drag it to a new value Use the mouse to click the up and down arrows or press the arrow keys on your keyboard to increase or decrease the values in small increments BD FACSAria User s Guide Sorting Controls All sorting on the BD FACSAria instru
92. e Instrument Quality Control on page 105 e Sample Optimization Using Instrument Setup on page 121 e Recording and Analyzing Data on page 129 99 Starting Up the Instrument Follow these steps to start up your BD FACSAria cell sorter 1 Turn on the instrument main power ensure that the lasers are off Tf the instrument was just shut down wait until the system is fully depressurized stops hissing before you turn on the main power A NOTICE Do not turn on the lasers until you have activated the stream Running the lasers without activating the stream can degrade the performance of the cuvette flow cell main power Main Power O Figure 4 1 Power panel 2 Start up the computer launch BD FACSDiVa software To start the software double click the application shortcut on the desktop 3 Verify fluidics levels in the Instrument frame replenish fluids or empty the waste if needed To display the Instrument frame click the Instrument button in the Workspace toolbar HE Verify fluid levels at the bottom of the frame sheath waste DI bleach ethanol Instrument Connected E A t 100 BD FACSAria User s Guide To service the fluid containers see Setting Up the Fluidics Cart on page 102 and Emptying the Waste on page 104 From the BD FACSDiVa Instrument menu choose Fluidics Startup click OK at the prompt Startup Status G Fluidics is starting You will be notified when the system is ready The Fl
93. e Select the Populations tab and deselect the checkboxes for Events and Parent REdit Statistics View SS e Set up the Statistics tab to display the mean and CV for FSC and each fluorescence A channel RJEdit Statistics View e Verify that Decimal Places is set to 1 for the CVs and 0 for the means e Click OK 7 Create other templates for the red and violet lasers 110 BD FACSAria User s Guide 8 Create appropriate plots and Statistics views on the Red and Violet templates respectively For example create the following plots for the red laser e FSC vs SSC dot plot e APC A APC H and APC Cy7 A histograms An example of a Red laser template is shown in the following figure 50 021703 Red 100 150 FSC A 200 250 1 000 021703 Red Count 50 100 150 200 250 APC A x 1 000 Count 50 021703 Red 100 150 APC Cy7 A 200 250 x 1 000 021703 Red 50 100 150 200 250 APC H 1 000 Experiment Name Tube Name Population FSC A Mean FSC A cv Feb 03 Red APC A APC A APC Cy7 A APC Cy A Mean cy Mean cy Call Events RHE HH HEO AAC OHR A Chapter 4 Running Samples 111 Verifying Area Scaling and Laser Delay Since the stream position is fixed and the laser paths are aligned on the stream laser signal s
94. e the folder with your name Alternatively you can name the folder Instrument OC or you can create an Instrument QC folder inside another folder Refer to the BD FACSDiVa Software User s Guide for ideas on how to organize Experiments Mi Tip To place an Experiment inside a folder select the folder before you create the Experiment 5 Press Ctrl E to create a new Experiment rename the Experiment with an appropriate name For example use the current month and year Instrument QC or the operator s initials followed by an appropriate identifier 106 BD FACSAria User s Guide 6 Rename the new Specimen with today s date rename the first Tube Blue This Tube will be used to measure signals from the blue laser 7 Select the Blue Tube in the Browser and click on the Instr Settings gt Parameters tab in the Inspector 8 Deselect the Log checkboxes for all fluorescent parameters and select the Height checkbox for any fluorescent parameter When you select one checkbox for Height H the checkboxes for all other fluorescent parameters are also selected he Inspector xj Tube Labels Acq Instr Settings Keywords Parameters Threshold Compensation Ratio Parameter voltage Log a _H w FSC 250 DIB Pp p ssc 300 peoo FITC 500 OlMiMio r PE 500 OlMiMio r PE Texas Red 500 DIM M b PercP Cy5 5 500 OlMiMio PE Cy7 500 DIPRIB IO APC 500 aaa 9 Click the N
95. ease the Purity Mask Erratic sort rate Flow Rate too high Decrease the Flow Rate Unexpected sort results Incorrect drop delay Reset the drop delay See Determining the Drop Delay on page 145 Incorrect Sort Precision mode Verify that the Sort Precision mode is appropriate for your sorting requirements See Sort Precision Modes on page 73 Sweet Spot off Keep the Sweet Spot on during sorting Drop 1 changed after setting drop delay Reset the drop delay each time you change the Drop 1 value See Determining the Drop Delay on page 145 Laser delay changed after setting drop delay Reset the drop delay each time you change the laser delay See Determining the Drop Delay on page 145 Incorrect logic in Population Hierarchy Verify the gating strategy 202 BD FACSAria User s Guide Sorting Troubleshooting continued Observation Possible Causes Unexpected sort results Sorting parent and child continued populations into two different tubes Recommended Solutions If you try to sort a parent and its child population into two tubes BD FACSDiVa software ignores the child events in both tubes Create a new subset under the parent population consisting of NOT Child Sort the child population into one tube and the NOT Child population into another tube Acquisition Troubleshooting Observation Possible Causes No events in plots after Acquisition pointe
96. eck the filter surface for streaks Inspect a quarter inch diameter section in the center of the filter for scratches Filters are coated with different dielectrics that can get scratched If you see scratches replace the filter Insert the cleaned filter into the octagon or trigon Make sure the filters are pushed all the way in 192 BD FACSAria User s Guide 7 Troubleshooting The tips in this section are designed to help you troubleshoot your experiments Additional troubleshooting information can be found in the BD FACSDiVa Software User s Guide If additional assistance is required contact your local BD Biosciences technical support representative See Technical Assistance on page xi Troubleshooting suggestions in this chapter are grouped under the following headings Troubleshooting the Stream on page 194 Troubleshooting the Breakoff on page 198 Sorting Troubleshooting on page 199 Acquisition Troubleshooting on page 203 Fluidics Cart Troubleshooting on page 209 Electronics Troubleshooting on page 210 193 Troubleshooting the Stream Observation Possible Causes Stream not in center Difference in keyed stream of aspirator position between nozzles Recommended Solutions If you have just changed the nozzle use an Allen wrench to loosen the screws on either side of the sort block Adjust the angle of the sort block until the stream flows into the center of the waste aspirator and then tighten the
97. end of the drop For example when the Yield Mask is set to 16 and an event is within 8 32 from the beginning of a drop the previous leading drop will be sorted If an event is within 8 32 from the end of a drop the following trailing drop will be sorted See Figure 2 18 on page 70 Chapter 2 Theory of Operation 69 70 trailing drop sorted drop being interrogated leading drop not sorted A ii Nx Yield Mask target particle Figure 2 18 Target particle within a Yield Mask of 16 Tf the Yield Mask were set to 8 for the same target particle the target particle would fall outside of the Yield Mask thus no additional drops would be sorted See Figure 2 19 trailing drop not sorted drop being interrogated leading drop not sorted A DU Figure 2 19 Target particle outside a Yield Mask of 8 ay Yield Mask target particle NE Yield Mask When the Yield Mask is set to zero only one drop the drop containing the target particle will be deflected when the mask is set to 32 two drops will always be deflected Yield Masks between 0 32 will sort either one or two drops NOTICE When more than one drop is deflected in the same direction residual charge from the first drop degrades the quality of the side streams Thus when four way sorting or sorting into small wells where precise deflection is required
98. enerates 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 History Revision Date Change Made 333608 1 03 Initial release Contents About This Guide ix CONVENTIONS Li A a E RI dos x Technical Assistance ads bs xi Safety and Limitations xiii Taser Satety a SAS tate gate bates e a Gund Ges ove xiii Laser Classification 2 0 0 cece cece eee n ence eens xiv Precautions for Safe Operation 0 0c eee cee eens xiv Electrical Safety tot os 2eee Woh de do st ota gd Sea el oe xv Biological Safety it ote E T ae xvi General Safety ivi A ew AS RE wd gE Se ade da xvil Precaution Labels acia o a ia xvil Limitations eiii pee he ee aa XIX Chapter 1 BD FACSAria Instrument Components 21 Fluidics Cart 2 064085 Sa dea ade ks 22 Containers and Connectors 0 0 cece ee cence eee eens 23
99. ent for Accudrop optimization Because no data is recorded the Experiment can be reused as often as you like 1 Create a new Experiment and rename it Accudrop 2 Optional Rename the first Tube Accudrop Beads 3 Create an FSC histogram 4 Draw an Interval gate that encloses the entire histogram Set the endpoints of the interval at 26 and 262 143 A NOTICE For an accurate setting ensure that the histogram region encompasses the entire histogram 5 Create a Sort Layout 6 Set the Precision mode to Initial and the Target Events to Continuous Chapter 5 Sorting 145 AccuDrop Beads Sort Layout_001 Eg Device Precision Target Events Save Conflicts fb Tube b finitian y Continuous y mj Left EX P1 Continuous Sort Rate NA Confl Cnt NA Confl Rate NA Efficiency NA BN Sort w Pause a view Counters Sorting Beads to Determine the Drop Delay 1 Load a tube filled with a dilute suspension of Accudrop beads approximately 2 drops of beads in 0 5 mL PBS 2 Inthe Laser tab set the Window Extension to zero 3 Adjust the Flow Rate to achieve an event rate of 2 000 4 000 events per second NOTICE If this cannot be achieved using a Flow Rate setting between 1 and 5 adjust the bead concentration 4 Turn on the Voltage in the Side Stream window click Sort in the Sort Layout view 5 Click Cancel at the prompt to open the aspirator drawer Lit Confirm Q Aspirato
100. ents The main power button turns on the instrument and fluidics cart and enables the laser power buttons To turn on a laser press the corresponding button The button lights up when the power is on A notice Do not turn on the lasers until you have activated the stream Running the lasers without activating the stream can degrade the performance of the cuvette flow cell main power Main Power power cord circuit breaker Figure 1 18 Flow cytometer power panel The instrument circuit breaker is located next to the power cord Figure 1 18 The switch will need to be reset if there is a power surge in the laboratory 42 BD FACSAria User s Guide Emergency Stop Button The Emergency stop button to the right of the loading port Figure 1 19 is a safety feature that can be used to halt the movement of the loading port and ACDU stage in case of need The button lights up when a tube is loading to remind you to keep your hands away from the loading port Emergency stop button Figure 1 19 Emergency stop button The following occur when this button is pushed e The tube is unloaded from the sample injection chamber e The ACDU stage if in use stops moving e The stream is turned off e The deflection plate voltage is turned off e The aspirator drawer if open closes to protect sort collection tubes e A warning message appears on the screen Do not reset the button until the message appears To do so turn the button clockwi
101. ents To Display 500 evt D Flow Rate 1 0 El 112 BD FACSAria User s Guide Adjust the FSC gain and the SSC voltage to place the particles on scale in the FSC vs SSC dot plot e Select the Blue Tube in the Browser e Click the Instr Settings tab in the Inspector and then click the Parameters tab e Click the up and down arrows or drag the sliders to adjust the values Draw a region around the singlet population 020203 Blue 2 S a E oO ao a 50 100 150 200 250 50 100 150 200 250 FSC A 1 000 Select all histograms on the Blue template and select the P1 checkbox in the Inspector The histograms change to display data from only the Singlet population Adjust the FITC voltage to place the FITC H signal at approximately 100 x 103 Verify area scaling for the blue laser and adjust it if needed The FITC A intensity should match that in the FITC H plot To adjust the intensity click the Laser tab in the Instrument frame select the current Area Scaling value and enter a new value View the result of your change in the histogram plots See Figure 4 3 on page 114 for an example Chapter 4 Running Samples 113 Instrument Lx Instrument Lx Status Parameters Threshold Status Parameters Threshold Compensation Ratio Laser Compensation Ratio Laser Name Delay Area Scaling Blue 0 00 Red 37 20 0 95 Red 37 20 0 95 Violet 32 80 E 0 76 Violet 32 80 E 0
102. erated under these conditions the instrument poses no danger of exposure to hazardous laser radiation xiv BD FACSAria User s Guide Electrical Safety A WARNING Lethal electrical hazards are present in some lasers particularly in laser power supplies Many portions of the electrical system including the printed circuit boards are at a dangerous voltage level To prevent shock injury follow these guidelines e Connect the equipment only to an approved power source Do not use extension cords Have an electrician immediately replace any damaged cords plugs or cables e Do not remove the grounding prong from the power plug Have a qualified electrician replace any ungrounded receptacles with properly grounded receptacles in accordance with the local electrical code e Do not perform instrument maintenance or service procedures except as specifically stated in the user s guide A WARNING A 12 000 volt potential exists between the deflection plates when they are on Contact with the charged plates results in serious electrical shock Do not touch the deflection plates when the voltage warning light is illuminated or when the software indicates that the plate voltage is on The plates remain energized even when the sort block door is opened A CAUTION The nozzle and the stream charging wire conduct electrical charge during sorting To prevent shock do not touch the nozzle or the stream charging wire when the voltage warning light
103. erates an image used to monitor drop formation It is focused on the stream just below the nozzle to provide an image of the drop breakoff e The lower camera generates an image used for the BD AccuDrop function It enhances the ability to see side streams and assists in setting an accurate drop delay value US Patent No 6 372 506 BD FACSAria User s Guide upper e A ZA E camera O 3 Ps EA X y TT f pol o A a 7A T 7 o F ra A y AA S diode laser micrometer dial lower camera viewing window Figure 1 17 Stream viewing optics Special image processing software allows you to view the stream images from each camera within separate windows in BD FACSDiVa software See Sorting on page 60 for more information about viewing the streams and to learn how BD FACS Accudrop components are used to determine the drop delay Instrument Electronics The electronic components consist of power controls and connectors along with processing boards in the card cage This section describes only user adjustable instrument electronics for more information about the electronics see Signal Detection on page 51 Chapter 1 BD FACSAria Instrument Components 41 Power Panel Power to the instrument lasers and fluidics cart is supplied by a power cord plugged directly into a standard electrical outlet there are no special facilities requirem
104. est peak in 556286 DNA QC Particles kit BD Biosciences 349523 216 BD FACSAria User s Guide Reagents Reagent Supplier Catalog No BD FACSFlow sheath fluid BD Biosciences 340398 877 232 8995 US and Latin America 342003 other countries BD FACSRinse detergent BD Biosciences 340346 BD FACSClean solution BD Biosciences 34xxxx Ethanol Various Chlorine bleach 5 sodium Clorox or other major supplier hypochlorite to ensure that the bleach is at the correct concentration and free of particulate matter Monoclonal antibodies BD Biosciences A Dyes and fluorochromes Molecular Probes 800 438 2209 Sigma 800 325 3010 BD FACS Lysing Solution BD Biosciences 349202 a Refer to the BD Biosciences Immunocytometry Products Catalog or the BD Biosciences website www bdbiosciences com b US Patent Nos 4 654 312 4 902 613 5 098 849 Appendix A Supplies and Options 217 Labware Item 1 mL microtubes Supplier Bio Rad Laboratories 800 424 6723 Catalog No 223 9391 1 000 per box 5 mL polystyrene test tubes 12 x 75 mm BD Falcon BD Biosciences 877 232 8995 e uncapped 125 per bag e 352052 e capped 125 per bag e 352054 e capped 25 per bag e 352058 e with cell strainer cap e 352235 25 per bag 15 mL conical centrifuge tubes BD Biosciences BD Falcon 877 232 8995 Polypropylene 50 bag e 352196 Polyprop
105. et clala visjela 2 3 Experiments My Folder f My Experiment 8S Instr Settings Instrument Connected MW El A x 200 20 Instr Settings Kewwords 1 000 Tube Labels Acq Events to Record 30000et y Stopping Gate Al Events Y Storage Gate Al Events y Tube_001 Threshold Rate Threshold Count 51000 evt Electronic Abort Rate Electronic Abort Count Processed Events 49000 evt Elapsed Time Tube_001 Oevtis 00 00 05 Next mm oad E Acquire E Record Storage Gate All Events V Stopping Gate All Events y Events To Record 30000 evt vw Events To Display 5000 evt fadl Flow Rate 1 0 El Figure 3 1 BD FACSDiVa workspace O Click a button in the Workspace toolbar to hide or show the corresponding frame Frames can be resized by dragging a border or corner O The Browser frame lists all experimental data in a hierarchical view provides an interface for setting up Experiments and contains an Acquisition pointer control for acquiring and recording data 78 BD FACSAria User s Guide O When connected to an instrument the Instrument frame shows instrument Tube and Laser controls connectivity status fluidics level indicators sheath waste bleach deionized water ethanol status messages instrument settings for the current acquisition The W
106. eters about 58 adding 225 assigning 56 84 224 labels 130 measuring 58 107 scatter distorted 207 pausing sorting 62 95 152 phase 66 Phase Masks about 72 Yield Masks using with 70 72 photodiodes 56 photomultiplier tubes PMTs about 39 56 applying voltages 58 124 assigning 56 84 224 plate voltage 66 plates installing 154 sorting into 153 plenum about 28 cleaning 175 plots creating 109 excessive debris 207 no events in 203 unexpected events in 205 Population Hierarchy 115 234 BD FACSAria User s Guide populations sorting 68 93 137 149 troubleshooting 201 208 power buttons 42 cord xv fluidics 23 fluidics cart 25 instrument 42 laser 42 precaution labels xvii Precision Modes 69 73 See also Sort Precision modes preferences user 106 136 pressure air 22 25 sample 48 sheath 46 81 priming fluids 104 172 printing instrument settings 116 Sort Reports 98 templates 116 purging filters 182 Purity Masks 71 Purity mode 73 149 Q quality control QC 105 R recording compensation Tubes 127 data 115 129 131 during sorting 151 refilling containers 103 removing ferrules 185 filters 183 223 reports instrument status 85 printing 98 116 sort 97 results troubleshooting 202 reusing analyses 135 rotating sort block 32 141 S safety biological xvi electrical xv general xvii 43 laser xiii sample agitation 81 core diameter 48 flow 47 injection chamber 29 inter
107. evere problem such as a clog is detected by the Sweet Spot the stream is shut off and sorting is stopped the deflection plates high Stream Ll Sweet Spot UN target value shut off the aspirator drawer closes and the sample tube is unloaded BD FACSAria User s Guide ES Gap actual value Preprogrammed values can be downloaded to the Breakoff window by choosing High Medium Low or Custom from the Sort gt Sort Setup menu Note that changes to values in the Sort Setup windows Breakoff and Side Stream are automatically saved At startup the last settings used on the instrument are restored except the Stream and Sweet Spot controls which always default to off Default values can be restored by choosing Sort gt Sort Setup gt Restore Defaults For more information see Sort Setup on page 89 Table 2 1 Breakoff window controls Control Stream button Description El and off Ef by clicking ion x Sweet Spot button Stream Enables automatic adjustment of the drop drive E Sweet Spot amplitude to maintain the stability of the breakoff point When the Sweet Spot is on the Amplitude and Frequency fields are disabled the amplitude is automatically adjusted by the software To enable the fields turn off the Sweet Spot Amplitude field Adjusts the amplitude or intensity of the drop drive from 1 0 80 0 volts The drop drive amplitude determines the breakoff point A higher
108. ext button in the Acquisition Controls frame to create an additional Tube for each laser For example if you have a three laser system create a Red Tube and a Violet Tube Your Experiment should look similar to that shown in the following figure Feb 03 85 Instr Settings 2 3 Acquisition Template Template1 E A 021703 U Blue U Red U Violet Chapter 4 Running Samples 107 10 Use the Experiment Layout dialog box to set the Events to Record to 10 000 evt for all Tubes in the Experiment e Choose Experiment gt Experiment Layout and click the Acquisition tab e Select all three Tubes e Choose 10 000 from the Events to Record drop down menu IN Experiment Layout Labels Keywords Acquisition Events to Record f 10 000 y JE Feb 03 GF 021703 Blue U Red TJ Violet Setting Up an Acquisition Template Next you need to create plots to display data In BD FACSDiVa software plots can be set up on worksheets or Acquisition Templates Acquisition Templates are useful when you want to reuse the same set of plots to display data from multiple Tubes Refer to the BD FACSDiVa Software User s Guide for more information about Acquisition Templates This section describes how to set up three Acquisition Templates each one will be used to display data from one laser 108 BD FACSAria User s Guide 1 Create an Acquisition Template by selecting the Acquisition Template tool in the Browser
109. ey are on 3 Choose Instrument gt Cleaning Modes gt Prepare for Aseptic Sort A dialog box appears with instructions on how to set up for cleaning Y Confirm Py To prepare for aseptic sort do the following N 1 Replace sheath filter with bypass tubing 2 Unplug sheath tubing from sheath inlet 3 Unplug ethanol tubing from ethanol inlet and plug into sheath inlet 4 Install tube with ethanol on the loading port Cancel 178 BD FACSAria User s Guide 4 Remove the sheath filter by pressing the tabs on each quick disconnect coupling coupling coupling 5 Install bypass tubing in place of the sheath filter Push the tubing coupling into each quick disconnect port until you hear a click bypass tubing 6 Detach the sheath tank tubing and the ethanol tubing from their respective ports on the fluidics cart Figure 6 3 on page 180 Chapter 6 Shutdown and Maintenance 179 180 ethanol port sheath port Figure 6 3 Sheath and ethanol ports Plug the ethanol tank quick disconnect into the Sheath port Install a tube containing ethanol onto the loading port click OK A progress message appears During the next 3 5 minutes the instrument performs the following e primes all fluid lines with ethanol e turns on the stream e loads the ethanol tube and starts running it Once the instrument is running ethanol through all lines a message appears xi Ethanol is now running through the sheath
110. f the slide Make sure that you set up your Sort Layout accordingly See Figure 5 5 on page 155 154 BD FACSAria User s Guide A3 0010000000 42 0 O 000000O ANOJAOOO0O0O0DOO Sort Sample Sort Layout xj Device Precision Target Events Save Conflicts Slide Frosted End y sinate Cell X fa A esas CA oss E EA ea ET JET View Counters Figure 5 5 Sort order on a slide Setting Up the Stream This section describes how to optimize side stream deflection and how to adjust the Home location When sorting into a plate or onto a slide the stage is preprogrammed to move a set interval between wells on a plate or spots on a slide The Home location is used as the starting point the far left stream should hit the center of the well in the top left corner of a plate or the top left corner of a slide at the Home location Default Home location coordinates exist for each standard sort collection device Falcon multiwell plates with 6 24 48 96 and 384 wells and standard or frosted end slides For other plate types you will need to set the Home location See Creating a Custom Device on page 158 Use the following procedure to verify the Home location and adjust it if needed 1 Optimize the drop delay See Determining the Drop Delay on page 145 Chapter 5 Sorting 155 2 Turn on the deflection plates Click the Voltage control Q in the Side S
111. f your sample tube e If you are replacing the secondary sample line leave approximately 0 1 in 0 25 cm of tubing extending out of the ferrule on both ends This length is referred to as the pilot as shown in Figure 6 5 on page 186 Chapter 6 Shutdown and Maintenance 185 pilot male nut ferrule length ferrule tubing internal y y tubing pilot pilot male nut length depth internal Figure 6 5 Components of a compression fitting diagram courtesy of Valco Instruments Co Inc 6 Insert the pilot tubing into its fittings ensuring that the tubing reaches the intended pilot depths A NOTICE Try not to bend the primary sample line during insertion e For the primary sample line insert the long end into the sample injection chamber fitting Push the tubing from the top until it is slightly above the bottom of the chamber viewing window Hand tighten the nut on top of the chamber and then insert the short end into the HPLC valve e For the secondary sample line insert one end into the HPLC valve and the other end into the cuvette flow cell fitting A NOTICE Within the HPLC valve and the cuvette flow cell fitting make sure the pilot is seated flush against the pilot depth Dead volume between the pilot and the pilot depth can lead to sample carryover or leaking 7 Hand tighten each nut firmly NOTICE Do not overtighten the nut and do not use tools Over tightening the nut can kink or damage the tubing
112. field s corresponding to the tube s well s or spot s where the population will be sorted and choose a defined population from the Add menu After you click in a sort location field a menu appears where you can choose to add delete or clear all populations in the field Figure 3 7 z Sort Sample Sort Layout xl Device Precision Target Events Save Conflicts l Tube X y Continuous y B oO P1 Continuous P2 Sort Rate mne a Delete gt Confl Cnt Me Conf NA Clear All Mrs Efciency NA Sort mi Pause ja z view Counters Figure 3 7 Adding populations to be sorted After you add a population the population and the number of target events are written to the corresponding sort location field Mi Tip Select a row or column header to select all fields in that row or column After adding a population it will be written to all selected fields at once 6 Specify whether to save sort conflicts by selecting the Save Conflicts checkbox This checkbox is enabled only when using a two or four tube layout When selected all sort conflicts are sorted into a default location e For a two tube layout conflicts are sorted to the right no other populations can be sorted to the right most tube Chapter 3 Using BD FACSDiVa Software 93 Sort Sample Sort Layout Lx Device Precision _ ee Events Save Conflicts 2 Tube v ruiy O Purity ee Left Confli
113. ftware information needed for instrument setup For more detailed instructions on how to use the software refer to the BD FACSDiVa Software User s Guide The BD FACSAria 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 New users of the BD FACSAria flow cytometer should read e Chapter 1 to become familiar with your instrument components e Chapter 2 to understand how the instrument works and to learn about the software components used to control different subsystems e Chapter 3 to see where software components are located Instructions for routine acquisition analysis and sorting can be found in Chapters 4 and 5 Once you become familiar with routine operation and need only a quick reminder of the main steps use the quick reference guide located in the front pocket of this user s guide 1x Conventions The following tables list conventions used throughout this guide Table 1 lists the symbols that are used to alert you to a potential hazard The word accompanying the hazard symbol indicates the level of severity as shown in Table 2 Text and keyboard conventions are shown in Table 3 Table 1 Hazard icons Symbol Hazard Symbol Hazard A Dangerous voltage A Highly flammable A Laser radiation A General a Biological risk Table 2 Severity indicato
114. g any of the selection handles to change the size and shape of the gate 6 Select all fluorescence histograms on the Unstained Control worksheet 7 In the Plot Inspector select the Show Grid checkbox Figure 4 8 hal Inspector Ea Plot Title Labels Histogram Tube ser Unstained Control X Parameter FITC A Y Parameter F checkbox Background Color Population All Events M Er Figure 4 8 Plot Inspector for fluorescent plots In a four log display values are displayed from 26 262 143 Thus the first log decade ranges from 26 262 Gridlines are used to delineate log decades on plots Chapter 4 Running Samples 125 8 Optimize the voltages to place the negative population for each fluorescent parameter within the first log decade Figure 4 9 Unstained Control x 1 000 S5C A Count 50 100 150 200 250 Unstained Control FSC A 1 000 Unstained Control Count Unstained Control 10 10 10 10 PerCP Cy5 5 A Count Unstained Control 10 10t APC Cy A Figure 4 9 Unstained Control Tube after PMT adjustment 9 Click Record when all events have been recorded click Unload and remove the unstained control tube from the cytometer A NOTICE Do not change the PMT voltages after the first compensation Tube has been recorded In order to calculate compensation all Tubes must be recorded with the same PMT voltage settings If y
115. go BD CaliBRITE BD FACS BD FACSAria BD FACSDiVa BD FACSFlow BD FACSRinse and BD Falcon are trademarks of Becton Dickinson and Company Clorox is a registered trademark of The Clorox Company Fluoresbrite is a trademark of Polysciences Inc JDS Uniphase is a trademark of the JDS Uniphase Corporation Kimwipe is a registered trademark of the Kimberley Clark Corporation Microsoft and Windows are registered trademarks of Microsoft Corporation Q tip is a registered trademark of Chesebrough Pond s Inc Sapphire is a trademark and Coherent is a registered trademark of Coherent Inc SPHERO is a trademark of Spherotech Inc Texas Red and Alexa Fluor are registered trademarks of Molecular Probes Inc All other company and product names might be trademarks of the respective companies with which they are associated Guide written by Kim Gautho edited by Joan Miller produced by Pushpa MacFarlane Regulatory Information For Research Use Only Not for use in diagnostic procedures 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 NOTE 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 g
116. h reagents Do not use the Instrument Configuration dialog box 3 Name the configuration and click OK 4 Select the new configuration in the list of configurations click once on the Add Parameter button for each parameter to be added 5 Specify a laser and detector for each parameter enter parameter names in the corresponding Parameter fields Appendix A Supplies and Options 225 Use the drop down menus to choose lasers and detectors You must choose a detector before you can edit the Parameter field Your Laser selection determines the number of choices in the Detector menu ie the Blue laser allows seven detector choices while the Red laser allows only three Remember to order your fluorochromes from longest wavelength to shortest 6 Use the drop down menu at the top of the dialog box to assign the side scatter parameter You must assign SSC to ensure the parameter is excluded from the compensation matrix Once it has been assigned the SSC Parameter field cannot be edited Side Scatter Parameter Parameter Laser Detector FSC Blue FSC ssc Blue D FITC Blue C 7 To make the new configuration the current configuration click Set Configuration then click OK A NOTICE You must click Set Configuration for the new configuration to apply For accurate data results always verify that the instrument optics setup matches the current Instrument Configuration 226 BD FACSAria User s Guide Inde
117. he cuvette The strobe lens and upper camera window are located behind two circular openings on either side of the cuvette You will see the end of the swab in the Breakoff window when you intercept either opening upper camera window 6 Gently wipe the upper camera window and then the strobe lens opposite the window to remove any saline 7 Repeat with spectral grade methanol or absolute ethanol until clean Chapter 6 Shutdown and Maintenance 191 Reinstall the grounding plate nozzle and deflection plates See Cleaning the Nozzle and Grounding Plate on page 165 for details Adjust the plates so there is a gap of approximately 0 28 in 0 7 cm between them at the top Cleaning Optical Filters Optical filters should be inspected occasionally and cleaned as necessary The frequency will depend on how often the filters are handled NOTICE When cleaning or replacing a filter handle with care to avoid scratching the surface and to prevent the filter from falling out of the holder Use Q tips optical lens paper and spectral grade methanol or absolute ethanol in a dropper bottle do not use acetone to clean optical filters 1 Wrap a triangular section of the lens paper around the cotton end of a Q tip Moisten and seal the end with a few drops of alcohol Holding the Q tip in a horizontal position gently rub any spots on the filter surface and wipe clean Allow the solvent to evaporate and ch
118. he drop was deposited If you need to move the stage to the front close the Device Setup dialog box and click the Access Stage control in the Sort Layout view Wipe the collection device dry and place it back on the tray support If needed click the Access Stage control to send the stage back and choose Sort gt Home Position to access the Device Setup dialog again Adjust the Home location if necessary Click the appropriate Arrow buttons to move the tray support as needed Large arrows move the tray by five steps small arrows move the tray by one step Repeat steps 6 through 9 until the drop is centered appropriately Click Set Home then Close Click the Voltage control J to turn off the deflection plates Proceed with Sorting on page 149 Chapter 5 Sorting 157 Creating a Custom Device You can program the ACDU stage to sort into any grid configuration Create a custom device by entering the number of rows and columns and setting the Home and Farthest locations BD FACSDiVa software calculates the increment between rows and columns to determine the sort locations farthest p0 O O home Figure 5 6 Home and Farthest location on 9 drop slide example 1 Choose Sort gt Custom Devices 2 Click the Add button in the Custom Devices dialog box A new device is added to the list of custom devices By default devices are named Custom Device_00x where x is the next consecutively numbe
119. he nozzle or the stream charging wire when the voltage warning light is illuminated or any time during sorting Chapter 1 BD FACSAria Instrument Components 31 32 Sort Block After leaving the nozzle particles pass through the sort block where they are either transported to waste via the waste aspirator or sorted into a collection device in the sort collection chamber The sort block houses the high voltage deflection plates along with the aspirator and aspirator drawer Figure 1 11 adjustment screw aspirator aspirator drawer sort collection device Figure 1 11 Sort block with door open Note that the entire sort block assembly can be rotated on a fixed pivot point to adjust the position of the stream in the waste aspirator If the keyed stream position differs between an old nozzle and a new one the stream might not hit the center of the aspirator after the nozzle is changed In this case you can change the angle of the sort block by loosening the adjustment screws on either side of the deflection plates and rotating the sort block Tighten the screws when the stream is re centered in the aspirator BD FACSAria User s Guide Deflection Plates The high voltage deflection plates are used to deflect side streams during sorting The plates are turned on and off using the Voltage control in the Side Stream window see Side Stream Formation on page 65 A red warning light is
120. he target value and the actual value is acceptable Gap field Gap between the stream breakoff and the top of the first drop The default Gap setting is 6 pixels and in general it should never be adjusted Differences of 1 2 pixels between the target value and the instrument generated value are acceptable Typically when setting up for sorting you use the Amplitude to set the required drop breakoff and copy the generated Drop 1 value to the target field Then you turn on the Sweet Spot to maintain the drop breakoff When the Sweet Spot detects a gt 1 pixel difference between the target Gap setting and the actual Gap it adjusts the Amplitude to reduce the Gap When a gt 2 pixel difference is detected the Sweet Spot stops sorting temporarily stops charging the stream until the Gap is restored Once the Gap is back within range sorting automatically resumes Vv Tip To save screen space you can hide the fields at the bottom of the window after the amplitude has been set To do so right click the window and choose Hide Controls from the contextual menu You can also choose to hide button labels To show the controls again right click the window and choose Show Controls For information on setting the breakoff see Setting Up the Breakoff on page 141 BD FACSAria User s Guide Side Stream Formation Side streams are formed when the voltage is on and you are sorting or when you click Voltage then Test Sort in the Side
121. here are several instrument components that should be cleaned periodically or checked for wear and replaced if necessary See the indicated sections for the following maintenance procedures Procedure Recommended Frequency Cleaning a Nozzle on page 188 When stream irregularities indicate the nozzle is clogged Cleaning the Camera Windows on When smudges appear in the Breakoff or page 190 Side Stream windows Cleaning Optical Filters on page 192 As needed when changing a filter Cleaning a Nozzle Use one of the following methods to clean the nozzle when the stream appears blocked or distorted To verify that the nozzle is clogged examine the tip under a microscope Figure 6 6 shows an example of an unclogged nozzle tip O ring Figure 6 6 Magnified view of a nozzle tip b WARNING All biological specimens and materials coming into contact with them can transmit potentially fatal disease Handle nozzles as if capable of transmitting infection Wear suitable protective clothing and gloves 188 BD FACSAria User s Guide 8 9 Turn off the stream and lasers if they are on Remove the nozzle from the cuvette flow cell Pull out the stream charging wire and turn the nozzle locking lever 90 to the left Remove the nozzle by pulling it straight out Remove and set aside the O ring Sonicate the nozzle for approximately 1 minute Sonicate the nozzle in a test tube containing DI water or a mild detergent Repeat the sonicat
122. hould remain constant from day to day However different sheath pressures and nozzle sizes can impact the laser delay and area signal Each Sort Setup option inputs approximate Area Scaling and Laser Delay values for the corresponding sheath pressure You should verify the settings each time you change the sheath pressure and each time the nozzle is changed This section describes how to optimize area scaling and laser delay and how to optimize PMT voltages for the signal from each laser Optimizing Signal from the Blue Laser BD FACSDiVa software uses area as its default parameter The area measurement provides a complete measurement of the voltage pulse but it can be affected by the sheath pressure To ensure that each detector works within its linear dynamic range it is important to adjust the height and area measurements to the same magnitude Refer to the BD FACSDiVa Software User s Guide for more information about area scaling 1 Double click the Blue template icon in the Browser and verify that the green Acquisition pointer is in front of the Blue Tube 2 Install the tube containing fluorescent particles for the blue laser onto the loading port click Load in the Acquisition Controls frame 3 Set the Flow Rate to 1 0 HE Acquisition Controls x Blue 696 evtis 00 02 26 Next Bl Load B Acquire Record Restart Storage Gate O All Events id Stopping Gate O All Events Sai Events To Record 10000 evt bd Ev
123. http www bdbiosciences com Part No 333608 Rev A March 2003 BD FACSAria User s Guide BD Biosciences 2350 Qume Drive San Jose CA 95131 1807 USA Tel 877 232 8995 Fax 408 954 2347 Asia Pacific Tel 65 6 861 0633 Fax 65 6 860 1590 Europe Tel 32 53 720211 Fax 32 53 720450 Brazil Tel 55 11 5185 9995 Fax 55 11 5185 9895 Japan Nippon Becton Dickinson Company Ltd Tel 0120 8555 90 Canada Tel 888 259 0187 905 542 8028 Fax 905 542 9391 canada bd com Mexico Tel 52 55 5999 8296 Fax 52 55 5999 8288 O 2003 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 errors 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 the BD lo
124. ice list but is retained within any Sort Layouts where it was used 160 BD FACSAria User s Guide 6 Shutdown and Maintenance The BD FACSAria instrument is designed to require minimum maintenance However to preserve the reliability of the instrument you must regularly perform basic preventive maintenance procedures This chapter explains routine maintenance procedures you should follow to keep your instrument in good condition Maintenance procedures are presented as follows e Daily Shutdown on page 162 e Scheduled Maintenance on page 168 e Unscheduled Maintenance on page 188 161 Daily Shutdown Run the following procedures at the end of each day or when required Fluidics Shutdown on this page External Cleaning on page 164 Fluidics Shutdown At the end of each day the instrument is used run the Fluidics Shutdown procedure to remove sheath fluid from the sheath path and rinse all lines with DI water This prevents saline crystals from clogging the fluidics NOTICE Allow approximately 3 minutes to complete the shutdown procedure 1 2 3 4 Unload your sample tube if one is loaded Turn off the lasers Turn off the stream Choose Instrument gt Fluidics Shutdown The following message appears Shutdown Status G Fluidics is shutting down please wait Optional Click OK to dismiss the message Empty the waste and refill the DI water if prompted to do so See Setting Up the Fluidi
125. ices The sort collection chamber door should be kept closed when sorting into a plate The door keeps the chamber free of dust and other air borne particles and seals the chamber during aerosol evacuation for instruments equipped with the AMO Chapter 1 BD FACSAria Instrument Components 35 36 Optics System The BD FACSAria cytometer uses innovative designs for both the excitation optics and collection optics The optics can be viewed by opening the optics access door and the flow cell access door See the following sections for more information A NOTICE The flow cell access door is equipped with a shutter mechanism that shuts off the laser light when the door is opened To ensure there is no interruption to data acquisition do not open the door while sorting or recording Excitation Optics The excitation optics consist of lasers fiber optic cables beam shaping prisms and an acromatic focusing lens as shown in Figure 1 15 on page 37 For information about how signals are generated see Signal Generation on page 49 The BD FACSAria instrument uses low powered air cooled and solid state lasers that do not have special power and cooling requirements The instrument comes standard with 488 nm and 633 nm lasers and a third 407 nm laser is available as an option Lasa Wavelength Min Power Commonly Used nm mW Fluorochromes Coherent Sapphire 488 blue 13 FITC PE PE Texas Red Solid State PerCP PerCP Cy5 5
126. ich particles will be sorted into each sort location Sorting controls and counters are also displayed in the window See Sort Layout on page 90 Chapter 3 Using BD FACSDiVa Software 87 88 The Breakoff window displays an image of the stream and contains controls to adjust drop formation See Drop Formation on page 61 The Side Stream window displays an image of the side streams and contains controls to adjust electrical charges and the drop delay See Side Stream Formation on page 65 Sort Menu Choose commands in the Sort menu for the following Sort Setup e Sort Setup downloads the most recently used settings Sort Precision for high medium or low pressure sorting see Sort NE Setup on page 89 C Gremeort Laycut Home Device Custom Devices Sor Report Sort Precision opens a dialog box where you can choose or define a Sort Precision mode for handling sorting conflicts see Conflict Resolution During Sorting on page 69 New Sort Layout opens the default 2 Tube Sort Layout view where other Sort Layouts can be chosen see Sort Layout on page 90 Note that clicking the Sort Layout button on the Browser toolbar performs the same function Open Sort Layout opens an existing Sort Layout A Sort Layout must be selected in the Browser for this menu command to be enabled Alternatively double click any Sort Layout to open it Home Device opens a dialog box cont
127. iner through the large cap without any interruption to your experiment but if you detach any lines you will need to prime the system 1 Optional Disconnect the container s sensor and quick release connector if you need to move the container Figure 4 2 If you do not need to move the container skip to step 2 quick release connector sensor large sized cap Figure 4 2 Fluidics container lines Remove the large sized cap from the container Figure 4 2 Fill the container with the fluid indicated on the container label Replace the container cap and hand tighten it until it is fully closed uu F amp F U N Reconnect the sensor and quick release connector to their respective ports if needed Connect the lines if they were disconnected in step 1 see Figure 4 2 A NOTICE To ensure that the appropriate solutions are dispensed do not switch tank positions Make sure the label on each container matches the labelled port on the fluidics cart Chapter 4 Running Samples 103 6 Prime the fluidics system if you disconnected the fluid lines in step 1 From the BD FACSDiVa main menu choose Instrument gt Cleaning Modes gt Prime After Tank Refill Emptying the Waste b WARNING All biological specimens and materials coming into contact with them can transmit potentially fatal disease To prevent exposure to biohazardous agents expose waste container contents to bleach 10 of total volume before disposal Dispose
128. ing Accudrop beads in Initial mode In the Sort Layout view change the Precision mode to Fine Tune Chapter 5 Sorting 147 10 Optimize the drop delay Adjust the Drop Delay value in 0 03 drop increments click arrow control until the left side stream intensity is 290 Wait a few seconds after each click for a complete response to the Delay change Voltage Center Plate Voltage 4 000 2nd Drop 20H 3rd Drop 10H 4th Drop oat Phase 041 Figure 5 2 Sorting Accudrop beads in Fine Tune mode 11 Click the Optical Filter control to move the emission filter away from the camera 12 Reset the Window Extension to its original setting typically 2 13 Turn off the deflection plates 148 BD FACSAria User s Guide Sorting Before beginning the sort do the following e Perform the steps outlined in Setting Up for Sorting on page 138 e Use gating tools and subsetting methods to define the population s of Interest NOTICE Snap To gates cannot be used for sort gates Examples of gating analysis can be found in Analyzing Data on page 132 and in the BD FACSDiVa Software User s Guide Setting Up the Experiment M Tip If you are sorting into four tubes create a Four Way Purity Sort Precision mode See Precision Mode Four Way Purity Defining New Precision Modes on page 75 Yield Mask o Purity Mask 32 When more than one drop is deflected in the same Phase Mask 0 direction residual
129. ing the sort location field s right click the selected fields to see the menu e Enter the number of Target Events and the population s for the remaining sort location fields as needed e To display fewer counters in the Sort Layout window click the View Counters button and deselect a menu option The corresponding counter is hidden Only counters with a checkmark next to the name are displayed 150 BD FACSAria User s Guide Starting and Monitoring the Sort 1 2 3 Open the sort collection chamber door and install the collection tubes plate or slide Install the sample tube on the loading port and click Load Set the required Flow Rate Vv Tip Sorting results are optimized at lower flow rates lt 8 0 4 Optional Turn on the deflection plates and open the aspirator drawer The deflection plates turn off automatically each time a tube is unloaded If you do not turn them back on before beginning a sort a dialog box appears where you can turn on the plates and open the aspirator drawer by clicking OK Verify that the green Acquisition pointer is indicating the appropriate Tube in the Browser click Sort Click OK if you are prompted to open the aspirator drawer or turn on the deflection plates Lit Confirm 2 Aspirator drawer is in WASTE position and deflection plates are OFF The drawer must be in SORT position and plates must be turned ON in order to sort Click OK to move drawer to SORT position a
130. ion Chapter 2 Theory of Operation 67 68 Drop Charging Drops are charged when an event is detected that meets the defined sorting criteria as determined by the Sort Layout window The Sort Layout is a floating window containing all sorting instructions and controls Figure 2 17 The Sort Layout designates which device will be used to collect sorted particles and which particles will be sorted into each sort location Up to four sort counters can be displayed in the window to give ongoing status during a sort See Sort Layout on page 90 for more information about the Sort Layout window 35 psi_001 4way rectangle x Device Precision Target Events Save Conflicts Ja Tube gt frors2 z D Far Left Right FarRight mr 251744 P3 264697 P4 268980 P5 259199 Sort Rate 960 evtis 1010 evtis 1026 evtis 989 evtis Conf Cnt 101696evt 107034evt 109057 evt 104484 evt Conf Rate te l 408 evtis 416 evtis 398 evtis Efficiency 71 71 71 E Sort wrasse View Counters Figure 2 17 Sort Layout for four way sort Target events are identified by drawing gates around populations of interest in plots The Sort Layout window specifies which gated population should be sorted into each sort collection tube or spot in a plate or on a slide During sorting when an event is identified within one of the sort gates the drop containing the particle of interest is charged via the strea
131. ion as needed until the nozzle is clean Thoroughly dry the nozzle Carefully spray the nozzle with compressed air or wipe it gently with optical lens paper If you use lens paper make sure no fibers are left behind Use the wooden end of a swab to reinstall the O ring on the nozzle tip Carefully reinsert the nozzle into the flow cell Insert it at a slight downward angle to avoid contact between the O ring and the cuvette Push it gently all the way forward without rocking it from side to side Turn the nozzle locking lever clockwise to the 12 00 position and plug the stream charging wire into the nozzle Turn on the stream and make sure it flows through the nozzle properly Turn on the lasers Note that after reinstalling the nozzle you might need to change the angle of the sort block to re center the stream in the aspirator To do so loosen the adjustment screws on either side of the deflection plates and rotate the sort block see Figure 1 11 on page 32 Tighten the screws when the stream is centered in the aspirator For further assistance see Troubleshooting the Stream on page 194 Chapter 6 Shutdown and Maintenance 189 Cleaning the Camera Windows e Clean the lower camera window and the diode laser window when you have trouble viewing the side streams or you cannot set the drop delay using Accudrop Wipe the windows with a soft lint free cloth soaked with DI water and then dry the windows diode laser window
132. is illuminated or any time during sorting Safety and Limitations xv Biological Safety b WARNING All biological specimens and materials coming into contact with them can transmit potentially fatal disease To prevent exposure to biohazardous agents follow these guidelines Handle all biological specimens and materials as if capable of transmitting infection Dispose of waste using proper precautions and in accordance with local regulations Never pipette by mouth Wear suitable protective clothing eyewear and gloves Expose waste container contents to bleach 10 of total volume before disposal Dispose of waste in accordance with local regulations Use proper precaution and wear suitable protective clothing eyewear and gloves Prevent waste overflow by emptying the waste container daily or whenever the waste indicator shows the waste is getting full WARNING Cell sorters that use droplet generation methods like the BD FACSAria can produce aerosols around the sample stream When acquiring biohazardous samples follow universal precautions at all times Keep the sort block door closed during sorting If you need to access the sort block turn off the stream before opening the door xvi For information on laboratory safety refer to the following guidelines NCCLS documents can be ordered online at www nccls org Schmid I Nicholson JKA Giorgi JV et al Biosafety guidelines for sorting of unfixed cells Cytometry 19
133. le injection chamber and tube holders A variety of tube holders are provided with the instrument to accommodate tubes from 15 mL centrifuge tubes to 1 0 mL microtubes Figure 1 8 For a list of compatible tubes see Labware on page 218 To load a tube install the appropriate size tube holder in the loading port and place a tube in the holder When the Load button is clicked in the software see Acquisition Controls on page 86 the loading port rises to enclose the tube within the chamber After a tube is loaded the Load button changes to Unload Use the Unload button to lower the loading port after data has been recorded After each tube is unloaded sheath fluid flushes the sample tubing inside and out to reduce Chapter 1 BD FACSAria Instrument Components 29 potential sample carryover Using a special cleaning mode the entire sample injection chamber can be decontaminated with ethanol See Clean Bulk Injection Chamber on page 176 for more information A CAUTION To prevent injury from moving parts keep your hands and clothing 30 away from the loading port when a tube is loading or unloading Do not place objects under the loading port Cuvette Flow Cell The cuvette flow cell is the heart of the BD FACSAria instrument Figure 1 9 Within the flow cell hydrodynamic focusing forces particles through the cuvette in a single file stream where laser light intercepts the stream at the sample interrogation point
134. lect The opposite surface facing away from the center should be coated with an anti reflective coating with a minimum reflection of lt 1 of the wavelength range you want the filter to reflect Note that filters must be installed in front of each PMT to block unwanted laser light Appendix A Supplies and Options 223 You can order a filter holder using the following part numbers Holder Part No Mirror holder 342971 Mirror retainer 345662 Mirror backup spring 334979 Filter holder 333814 Filter retainer 333817 For the dichroic carefully assembly the glass filter in the holder using the retaining spring Assemble the filter with the arrow pointing toward the center of the octagon or trigon The angle of the dichroic is critical to achieving optimal results Note that any time you modify a detector array you will need to create a new Instrument Configuration to identify which PMT identified by laser color and letter will detect the emitted light See Defining a New Configuration on page 224 for instructions Defining a New Configuration BD FACSDiVa software is provided with default configurations specific to your instrument additional custom configurations can be defined You can switch between predefined configurations by selecting the appropriate configuration name and then clicking Set Configuration Vv Tip Create one configuration that lists all the fluorochromes used in your laboratory
135. ll times Keep the sort block door closed during sorting If you need to access the sort block turn off the stream before opening the door deflection plates warning light sort block sort collection chamber Figure 1 13 Sort block with door closed Additional aerosol removal is provided by the BD Aerosol Management Option AMO The AMO is an optional device that evacuates the sort collection chamber during sorting The AMO is equipped with a 0 01 um pore ultra low penetrating air ULPA filter to trap aerosolized particles The AMO pump evacuates the sort collection chamber 16 times per minute in normal evacuation mode and 69 times per minute in rapid evacuation mode BD FACSAria User s Guide Sort Collection Chamber Collection devices are installed in the sort collection chamber to collect sorted samples during sorting The following collection devices are provided with the instrument to accommodate a variety of tube sizes e Four way 1 mL collection tube holder e Four way 12 x 75 mm collection tube holder e Two way 12 x 75 mm collection tube holder e Two way 15 mL collection tube holder An automated cell deposition unit ACDU that sorts into multiwell plates and onto microscope slides is available as an option BD Biosciences also offers a temperature control option to maintain the temperature of sorted samples during sorting four way 12x75 mm collection devices o plate loader Figure 1 14 Sort collection dev
136. m charging wire in the nozzle 1 O om BD FACSAria User s Guide The amount and type of charge determines where the drop will be sorted For example in a four way sort drops with the most charge will be deflected into the outer streams while drops with less charge will be deflected into the inner streams Conflict Resolution During Sorting During sorting the cytometer deflects drops based on the characteristics of the particles in each drop and where the user wants to deflect them Conflicts can occur depending on the type of target particle where the particle is located within a drop or whether the drop is free of contaminating particles BD FACSDiVa software accurately measures particle position to within 1 32 of a drop Mask settings determine how drops are deflected when sorting conflicts occur There are three Mask Precision Mode Pury y settings each of which addresses a different type of vield Mask 32 conflict These settings are combined to define sort Purtyask 82 Precision Modes each mode is made up of a set of ee z 2 masks Precision Modes are defined in the Sort Precision dialog box accessed from the Sort menu Add WEJEtE Close Yield Mask The Yield Mask setting defines how close to the edge of the drop in 1 32 drop increments a particle of interest can be located before sorting an additional drop Half of each Yield Mask setting defines an equal area at each
137. ment is controlled by BD FACSDiVa software Sorting controls are shown in Figure 3 4 R BD FACSDi a Software File Edit View Experiment Populations Worksheet Instrument ERE Help al FOR l AE y E kamm x Cota 2 0 lisme al M alela ASIA majma ma steam Ep sheett eal Sweet Spot ini Custom Devices Ea Sort Report T Name E S Experiments fal DNA PN AccuDrop S Instr Settings E 8 Acquisition Templates S Template La Analysis L 2 Template 1 Drop Delay Sort Layout _ Drop Delay Sort Di Precision Target Events Save Conflicts ACDU Sort Layc 5 A specimen _001 fotus fever _ W o a T Tube_oo1 Len Right amp H PN Electronic Noise Ml Notes 631755 fal PN DNA QC i PN Nile Red T o fal PN Poly Blue mcr mro AZ View Counters da vottage Y Test Sort K Optical Fiter Milf attenuation Drop Detay 41 93 St o AO AO HA A HA Voltage Center Plate Voltage 6 000 2ft 2nd Drop 20 3raDrop 10t 4tnDrop ojt Phase 0H rop 1 190 a 67 Figure 3 4 BD FACSDiVa sorting controls 1 The Sort menu provides access to Sort Setup and Sort Precision modes Sort Layouts Sort Reports and sort devices used with the automated cell deposition unit ACDU See Sort Menu on page 88 O The Sort Layout window designates which device will be used to collect sorted particles and wh
138. mpensation This section describes how to perform sample optimization using the Instrument Setup feature Instrument Setup can be used to automatically calculate compensation settings For more information about this feature refer to the BD FACSDiVa Software User s Guide If you are performing compensation manually not all steps will apply Chapter 4 Running Samples 121 Creating the Experiment This section describes how to verify the instrument configuration create a folder and Experiment specify the parameters for the assay and add compensation Tubes 1 Choose Instrument gt Instrument Configuration and verify the current configuration Make sure appropriate parameters are listed and that the lasers and detectors reflect the setup of the detector arrays A CAUTION For accurate data results the instrument optics setup must match the current Instrument Configuration 2 Click the corresponding Workspace tools to display the Browser Instrument Inspector Worksheet and Acquisition Controls frames as needed 3 Optional Press Ctrl N to add a new folder to the Browser rename the folder with your name Alternatively you can name the folder Sample Optimization or you can create a Sample Optimization folder inside another folder Mi Tip To place an Experiment inside a folder select the folder before creating the Experiment 4 Press Ctrl E to create a new Experiment rename the Experiment with an appropriate name
139. n Tube name Sort Layout name type of collection device and the date and time of printing e Sort settings Sort Setup values precision mode and masks definition e Acquisition counters Threshold count processed events count electronic conflicts count and elapsed time e Sort counters counter values per sort destination or total sort count if sorting sequentially e Sort Layout Population s sort count and target event count for each sort location field Lit Sort Report x File ube Tube_004 Device 2 Tube Sort Report ort Layout Sort Layout_001 Report Date 2002 09 24 at 12 54 56 Sort Settings Sort Setup High Phase Mask 0 Frequency 9000 Single Cell Off Amplitude 60 00 Sweet Spot Off Phase 0 00 Breakorr 230 Drop Delay 4400 Gap fi Attenuation Off Plates Voltage 6 000 00 Precision Purity Voltage Centering 0 Yield Mask 32 Sheath Pressure T000 Purity Mask 32 Side Stream Voltage o Far Left Len Right Far Right 80 00 60 00 60 00 80 00 Neighboring Drop Charge 1 2nd Jrd 4th 20 00 10 00 0 00 Chapter 3 Using BD FACSDiVa Software 97 The Sort Report window contains a File menu where you can choose to print or export the report Exported comma separated value CSV files can be opened with a spreadsheet application such as Microsoft Excel 98 BD FACSAria User s Guide 4 Running Samples The following topics are covered in this chapter e Starting Up the Instrument on page 100
140. n Controls Lx Blue 696 evt s 00 02 26 Next Bl Acquire Bl Record Restart Storage Gate L_ All Events w Stopping Gate Call Events a Events To Record 10000 evt vw Events To Display 500 evt ei a Flow Rate 1 0 St Figure 2 4 Load button and Flow Rate field in Acquisition Controls frame Note that the relatively longer sample tubing on the BD FACSAria instrument results in a different flow rate between cells and beads Thus absolute counting using BD TruCOUNT beads can give erroneous results Hydrodynamic Focusing In the flow cell pressurized sheath fluid surrounds the sample fluid to hydrodynamically focus the core stream of suspended cells into the center of the cuvette where the particles are intercepted by the laser beam The difference in pressure between the sheath fluid and the sample fluid can be used to vary the diameter of the sample core A lower difference results in a relatively narrow core stream while a higher difference results in a wider sample stream sheath flow sheath flow sheath flow sheath flow sample flow sample flow eN y EN i laser beams laser beams low flow rate high flow rate 48 BD FACSAria User s Guide Ideally you want the core stream at its minimum diameter so that cells pass through the laser beam in a single file stream However depending on your application a lower resolution might be acceptable in order to acquire the data more quickly For example
141. n as fluorescence Fluorescence is always a longer wavelength lower energy photon than the excitation wavelength The difference between the excitation wavelength and the emission wavelength is known as the Stokes shift Some fluorescent compounds such as PerCP exhibit a large Stokes shift absorbing blue light 488 nm and emitting red light 675 nm while other fluorochromes such as FITC have a smaller Stokes shift absorbing blue light and emitting green light 530 nm The emission spectra for some commonly used fluorochromes are shown in Figure 2 5 See Table 1 1 on page 40 for the corresponding detectors FITC Alexa Fluor 430 100 Pacific Blue RPE PI APC PerCP PerCP Cy5 5 PE Cy7 Normalized Intensity Hoechst q L 00 UN gt 600 700 800 Wavelength nm 0 400 5 Figure 2 5 Emission spectra of commonly used fluorochromes 50 BD FACSAria User s Guide Signal Detection From the cuvette flow cell scattered and fluorescent light is collected by the fluorescent objective lens The lens collects and focuses fluorescent light emitted at each of the laser focal points onto individual collection fibers These fibers transfer the emitted light to the individual detector arrays This section describes how laser light is detected and translated into signals that can be displayed in a plot Detector Arrays There are two types of detector arrays in the BD FACSAria flow cytometer an octagon and
142. n by another population Right click the plot and choose Order Populations by Count Chapter 7 Troubleshooting 205 Acquisition Troubleshooting continued Observation Possible Causes Unexpectedly high event Threshold channel too low rate Recommended Solutions Adjust the Threshold channel See Adjusting the Voltages and Threshold on page 124 Sample too concentrated Dilute the sample Event rate too high Decrease the Flow Rate in the Acquisition Controls frame Bubbles in flow cell Turn off the stream wait a few seconds and turn on the stream again Unexpectedly low event Sample not adequately mixed rate Increase the Sample Agitation rate See Sample Agitation on page 81 Threshold channel too high Adjust the Threshold channel See Adjusting the Voltages and Threshold on page 124 Sample too dilute Concentrate the sample Sample line clogged Perform a sample line backflush See Sample Line Backflush on page 170 If necessary change the sample line Sample line clogged or kinked Backflush the sample line See Sample Line Backflush on page 170 Look for visible kinks in the line If kinks are noted change the sample line See Changing the Sample Lines on page 184 Sample line installed incorrectly Verify the sample line installation See Changing the Sample Lines on page 184 Sample aggregates Filter the sample 206 BD FACSAria
143. n the Control key the first time you click the tool The tool remains selected until you press Esc or select another tool Chapter 4 Running Samples 127 FITC Stained Control FITC Stained Control SSC A amp 1 000 50 100 150 200 250 50 100 150 200 250 FSC A x 1 000 FITC A Figure 4 10 Gating the positive population 9 Double click the next Stained Control Tube in the Browser to locate the corresponding plots on the worksheet 10 Repeat steps 8 and 9 for the remaining compensation Tubes Once all regions have been set you are ready to calculate the compensation 11 Choose Instrument gt Instrument Setup gt Calculate Compensation If the calculation is successful a dialog box appears where you can enter a name for the compensation Setup 12 Enter a name for the compensation Setup click OK lit Single Stained Setup Compensation calculation has completed successfully Name j Color Expt 030503 Cancel Vv Tip To keep track of compensation Setups include the Experiment name date or both in the Setup name The named Setup is automatically linked to the Experiment s instrument settings M Tip Collapse the Compensation Specimen to save room in the Browser 128 BD FACSAria User s Guide NOTICE BD Biosciences recommends that you always visually and statistically inspect automatically calculated overlap values The means of the positive controls should be aligned with the means of the negative
144. nce of aerosols escaping Slide the holder into the slotted fittings below the sort aspirator drawer and close the sort collection chamber door Turn on the deflection plates Click the Voltage control in the Side Stream window The voltage warning light illuminates indicating that the plates are charged Test Sort Optical Filter PM Attenuation Drop Delay 43 76 l rr r Voltage Center T Plate Voltage 6 000 2l 2nd Drop 20H 3ra Drop 10 lt t 4th Drop 5 lt t Phase ot Chapter 5 Sorting 143 Make sure the center stream image does not move after the plates are turned on If the stream image is off center use the Voltage Center slider to re center the stream Major movement of the center stream could indicate that the plates or area around the plates needs cleaning See External Cleaning on page 164 4 Turn on Test Sort and optimize the side streams Adjust the voltage sliders to view the required number of streams If you cannot see a stream image or the image is dim adjust the micrometer dial on the diode laser to better view the streams Figure 1 17 on page 41 e For four way sorting adjust the four slider controls until you see four streams e For two way sorting turn off the outer streams by moving the outer slider controls all the way to the center Adjust the inner two slider controls until you see two streams 5 Adjust the 2nd 3rd and 4th Drop settings to tighten the center
145. nd filters The following illustrations show how the collection optics are configured for a two and three laser system Two Laser System This is the standard configuration for a two laser system PerCP Cy5 5 or Pl APC Cy7 APC PE Texas Red Lt Instrument Configuration Current Configuration 1 laser 3 color atera EEG Parameter Detector 3 laser 9 color FSC Blue FSC ssc Blue F FITC Blue E PE Blue D PE Texas Red Blue c PerCP Cy5 5 Blue B IPE Cy7 Blue A APC _ Red B APC Cy7 Red A Add Configuration Delete Configuration Set Configuration Add Parameter pelete Parameter OK Cancel 220 BD FACSAria User s Guide Three Laser System This is the standard configuration for a three laser system PerCP Cy5 5 or Pl Cascade Blue PE Texas Red Lit Instrument Configuration Current Configuration 3 laser color Side Scatter Parameter Blue F 2 laser 7 color Parameter Detector FSC Blue FSC a ssc Blue F FITC Blue E PE Blue D PE Texas Red Blue c PerCP Cy5 5 Blue B PE Cy7 Blue A APC Red B APC Cy7 Red A Violett Violet B Violet2 Violet A Pa Add Configuration Delete Configuration Set Configuration Add Parameter Delete Parameter Appendix A Supplies and Options 221 Custom Configuration Use the following worksheet to set up a custom configuration Configuration Name gt signal
146. nd turn plates ON Click Cancel to start sortin current state Cancel A NOTICE If you click Cancel at the message prompt sorting will begin with the deflection plates off and the drawer closed Thus sort populations will be identified and counted but no deflection or sorting will occur If you sort with the drawer closed events will be sorted to waste Mi Tip You can click Record to save data for the Tube Acquisition and sorting continue when the required number of events has been recorded Chapter 5 Sorting 151 Sorting continues until the required number of cells has been sorted Acquisition stops and the drawer closes when sorting is complete If the number of Target Events is set to Continuous sorting continues until you manually stop sorting by clicking the Acquire or Sort button Monitor the sort progress from the Sort Layout view The number of events sorted into each sort location appears in the corresponding field The sort rate and sort conflict rate are displayed in the corresponding counter fields 35 psi_001 4way rectangle x Device Precision Target Events Save Conflicts la Tube y fror32 mai y Far Left Right Far Right far 251744 P3 264697 P4 268980 P5 259199 r Sort Rate 960 evtis 1010 evtis 1026 evtis 989 evtis Confl Cnt 101696 evt 107034 evt 109057 evt 104484 evt Conf Rate 408 evtis ze cas 416 evtis 398 evtis Efficiency
147. ng sections for a description of these BD FACSAria functions e Fluid Movement on page 46 e Signal Generation on page 49 e Signal Detection on page 51 e Electronic Processing on page 57 e Sorting on page 60 45 Fluid Movement 46 The fluidics system is responsible for moving particles from the sample injection chamber through the cuvette flow cell for interrogation and then to waste or into a collection device during sorting Figure 2 1 The following sections describe the controls used to move fluids through the BD FACSAria fluidics system Sheath Flow Sample Flow Figure 2 1 Fluidic movement via the fluidics system Sheath Flow When you turn on the instrument the fluidic pumps start up The fluidics system is activated when you choose the Fluidics Startup command in BD FACSDiVa software During the fluidics startup procedure sheath fluid is pumped from the sheath container through a 0 22 um filter into the plenum reservoir The reservoir is pressurized to deliver sheath fluid to the cuvette flow cell at a constant pressure You can view the current sheath pressure using the Instrument gt Sheath Pressure command Figure 2 2 on page 47 BD FACSAria User s Guide i Sheath Pressure ox Sheath Pressure 70 00 atl Figure 2 2 Sheath pressure level After fluidics startup sheath flow is controlled using the Stream button in the Breakoff window which is labelled with the chosen sort pressure Figure 2 3
148. not acquisition sort counters freeze when the Pause button is clicked Click the Pause button again to continue sorting and to continue incrementing the sort counters e Access Stage when the ACDU stage is in the back brings the stage forward to put a plate on or take a plate off the stage When the stage is in the front sends the stage to the back and out of the way so you can install a collection tube holder e Move Drawer moves the aspirator drawer in or out depending on its current state The default state is in For more information see Aspirator Drawer on page 33 Using Counters Counters provide ongoing status during sorting the fields cannot be edited To display fewer counters in the Sort Layout window click the View Counters button and choose a menu option The corresponding counter is hidden Only counters with a checkmark next to the name are displayed Sort Sample Sort Layout Ea Device Precision Target Events Save Conflicts p Tube y Purity y y L eft L Sort Rate NA NA Confl Cnt NA NA Conf Rate NA NA Efficiency NA NA wm Sort E Pause alz v Sort Rate v Conflicts Count v Conflicts Rate v Efficiency Chapter 3 Using BD FACSDiVa Software 95 96 Counters display the following information e Sort Rate number of events second that met the sort criteria and were sorted e Conflict Count number of events that met the sort cri
149. ntaminating particles or the drop will not be sorted Chapter 2 Theory of Operation 71 Phase Mask Particles near the drop edge can affect the breakoff and alter the trajectory of the deflected drop The Phase Mask restricts drop deflection when an event is too close to the edge of a drop or when there are events close to the edge of adjacent drops A Phase Mask is used to improve counting accuracy and side stream quality at the expense of yield For example when the Phase Mask is set to 16 the drop being interrogated will be sorted only if the target particle falls outside the Phase Mask Figure 2 22 trailing drop drop sorted leading drop Phase Mask Z trailing drop drop not sorted leading drop L_ Phase Mask Figure 2 22 Sorted and unsorted drop with Phase Mask of 16 Decreasing the Phase Mask to 8 allows more drops to be sorted However because the target particle is closer to the edge of the drop there is more variability in drop trajectory Figure 2 23 trailing drop drop sorted leading drop Mi A N Y L Phase Mask Figure 2 23 Sorted drop with Phase Mask of 8 Mi Tip BD recommends using a Phase Mask of at least 8 when sorting single cells Phase Masks cannot be used in conjunction with Yield Masks Thus when the Phase Mask is gre
150. nting is required Mi Tip Select the Single Cell checkbox to obtain the highest quality side streams and the most accurate counts When the checkbox is selected drops containing two target events acceptable with a Purity Mask are discarded The Yield Mask is disabled when Single Cell is selected The remaining modes are used mainly during drop delay determination Initial mode is equivalent to the Yield mode but it is named differently as a reminder to use this as the initial mode when setting the drop delay using Accudrop e In Initial mode only the Yield Mask is used at its maximum value thus recovery and yield are optimized at the expense of purity e In Fine Tune mode all Masks are set to zero for deflecting the maximum number of drops This mode is used to fine tune the drop delay value See Determining the Drop Delay on page 145 for more information BD FACSAria User s Guide Defining New Precision Modes Default Precision modes cannot be edited however you can create new modes and then choose them from the Precision Mode drop down menu 1 Choose Sort gt Sort Precision click Add Precision Mode Puy 15 Yield Mask 32 Purity Mask 32 PhaseMask sO Single Cell m Add pelete Close The current sort mode is duplicated and the Mask fields are enabled Optional Change the name of the mode in the Precision Mode field Enter values for the Yield Purity and Phase Masks Click to select the Single Cell
151. ntrols 80 level indicators 83 100 power 25 shutdown 80 162 startup 46 80 101 system 46 fluidics cart about 22 power 23 setting up 102 troubleshooting 209 fluids containers 23 levels 83 100 movement 46 priming 104 172 recommended 102 refilling 103 fluorescence about 50 emission spectra 50 signal troubleshooting 205 folders adding 106 Index 231 frames about 78 87 acquisition controls 86 showing 106 See also views windows frequency drop drive 63 G Gap 62 64 gating compensation Tubes 127 data 115 132 general safety xvii 43 grounding plate cleaning 165 H hardware ACDU 153 hazards biological xvi electrical xv laser xiii mechanical xvii 43 heating samples 82 102 height parameters 58 hiding controls 64 65 holders collection tube 35 143 sample tube 29 Home Device 88 155 hydrodynamic focusing 48 immunophenotyping analysis 132 Experiment 129 Initial mode 74 Inspector frame 79 232 BD FACSAria User s Guide installing bypass tubing 179 collection tube holders 143 grounding plate 167 nozzle 140 nozzle lever 167 plates 154 sample tubes 29 slides 154 splash shield 153 instrument about 21 26 adding configurations 224 cleaning 164 168 configuration 56 84 224 controls 79 disconnect error 210 doors 26 electronics 41 fluidics 27 46 not responding 210 optics 36 options 219 power 42 QC particles 216 quality control 105 settings printing 116 shutting down 162
152. of waste in accordance with local regulations Use proper precaution and wear suitable protective clothing eyewear and gloves 1 Disconnect the waste container s sensor and quick release connector from their respective ports on the fluidics cart waste port waste tank 2 Remove the large sized cap from the container and dispose of the waste 3 Add approximately 1 L of bleach to the waste container 10 L container Add a sufficient amount so that 10 of the total waste volume is made up of bleach 4 Replace the container cap and hand tighten it until it is fully closed 5 Reconnect the sensor and quick release connector to their respective ports 104 BD FACSAria User s Guide Instrument Quality Control Before you run samples on the cytometer you will want to optimize PMT voltage settings and ensure that the Laser Delay and Area Scaling values are appropriate for your experiment This section describes how to set up a quality control QC Experiment how to verify the Laser Delay and Area Scaling values and how to record parameter means and CVs for a fluorescent bead For examples of fluorescent particles that can be used in this experiment see Instrument Setup Particles on page 216 Note that when the Experiment and QC sample are constant changes in the recorded means and CVs indicate variations in instrument performance over time QC data should be analyzed for trends over 30 60 runs NOTICE QC results are affecte
153. omplete Long Clean Status G Long clean is complete Before turning on the stream run Fluidics Startup at least once to remove trace decontaminant from the sheath path Refer to the user s guide for complete instructions Click OK to dismiss the completion message 7 Usea clean dry cloth to remove residual ethyl alcohol from the loading port 8 Before using the system choose Instrument gt Fluidics Startup Chapter 6 Shutdown and Maintenance 177 Prepare for Aseptic Sort Use the Prepare for Aseptic Sort command when you want to decontaminate the entire sheath path with ethanol from the fluid line starting at the sheath inlet port in the fluidics cart to the nozzle outlet For the most complete decontamination autoclave the sheath container and sheath probe and install a new sheath filter before sorting Note that the plenum reservoirs can also be removed and autoclaved or cleaned with ethanol before running this cleaning mode if required To do so run a Long Clean with bleach or ethanol as described in Long Clean on page 173 and then clean the plenum reservoirs as described on page 175 NOTICE This procedure can take 10 40 minutes depending on how long you run the stream to remove trace decontaminant Cleaning the Lines with Ethanol 1 Ensure fluid levels are adequate empty the waste container if it is full This procedure requires approximately 250 mL of ethanol 2 Turn off the lasers and the stream if th
154. ong Clean options Long Clean with Bleach Long Clean with Ethanol and Clean Bulk Injection Chamber Use the Bleach option to remove adhesive dyes such as PI acridine orange or thiazole orange or when you want to decontaminate the sample lines Use the Ethanol option to decontaminate the lines using ethanol You can substitute another cleaning solution for bleach or ethanol but to prevent foaming do not use a detergent containing solution The Clean Bulk Injection Chamber option is described on page 176 b WARNING All biological specimens and materials coming into contact with them can transmit potentially fatal disease Use 10 bleach to decontaminate the sheath path Flushing the lines with 10 bleach is the only procedure recommended by BD Biosciences for decontaminating the instrument NOTICE This procedure can take 10 40 minutes depending on how long you run the stream to remove trace decontaminant 1 Ensure fluid levels are adequate empty the waste container if it is full For example if you are performing a bleach cleaning procedure make sure the bleach tank is full The long clean requires approximately 90 mL of cleaning fluid and 110 mL of DI water 2 Turn off the lasers and the stream if they are on 3 Choose Instrument gt Cleaning Modes gt Long Clean If you have not already turned off the stream a message appears reminding you to do so 4 Select a Long Clean option click OK Chapter 6 Shutdown and Maintenan
155. ontrols 87 94 counters 95 into plates 153 into slides 153 into tubes 143 monitoring 96 152 pausing 62 95 152 populations 68 93 137 149 recording data 151 report 97 setup 138 starting 94 151 stopping 94 troubleshooting 199 troubleshooting 101 194 viewing 40 strobe lens cleaning 191 supplies instrument 212 Sweet Spot about 61 62 control 63 T tanks See containers Target Events 93 technical assistance xi temperature control option 35 219 sample 82 test sort 66 156 threshold adjusting 124 troubleshooting 208 toolbar workspace 78 splash shield installing 153 stage accessing 95 236 BD FACSAria User s Guide trigons about 38 39 51 default filters 40 220 221 troubleshooting ACDU 200 acquisition 203 breakoff 141 198 CVs 207 deflection 200 electronic aborts 208 electronics 210 event rate 206 fluidics cart 209 low Area signal 205 populations 201 208 scatter parameters 207 signals 205 208 sort conflict rate 202 sort counters 201 sort results 202 sorting 199 stream 101 194 threshold 208 Tubes adding Sort Layouts 92 compensation 123 label specific 123 tubes agitating 81 heating cooling 82 102 holders 29 35 loading 29 47 sorting into 35 143 U Unload button 86 unloading tubes 29 47 user preferences 106 136 V views Sort Layout 90 statistics 109 See also frames windows voltage adjusting PMT 58 124 controls 66 warning light 33 W waste aspir
156. orksheet frame provides an area where you can create Acquisition Templates or worksheets containing plots gates statistics and custom text for a description The Acquisition Controls frame contains controls used to acquire and record data Along with the general controls described in the BD FACSDiVa Software User s Guide the Acquisition Controls frame displays additional fluidics controls for the BD FACSAria instrument See Acquisition Controls on page 86 6 The Inspector frame provides an easy to use interface for viewing or modifying Browser the attributes of a single object or set of objects on the worksheet or in the The Acquisition Status frame provides ongoing status during acquisition or recording Instrument Controls Most BD FACSAria specific instrument controls are accessed through the Instrument menu Instrument controls on the Instrument menu are described in the following sections e Fluidics Controls on page 80 e Instrument Configuration on page 84 e Instrument Status Report on page 85 The Enable Test Features command is password Instrument Fluidics Startup Fluidics Shutdown Cleaning Modes Sheath Pressure Sample Agitation Sample Temperature Instrument Configuration Instrument Name Enable Test Features Instrument Status Report Instrument Setup protected It is used by BD Biosciences service engineers
157. ort location field for spot on slide I Bt cms Epi El pra cma BN Sort m Fause a View Counters Figure 3 6 Sort Layout for frosted slide Setting Up a Sort Layout NOTICE If an Acquisition Template is displayed in the Worksheet frame when you create a new Sort Layout the Sort Layout will be added to the Acquisition Template rather than the Tube To add a Sort Layout to a Tube switch to the Worksheet view and click on the Tube icon in the Browser before creating the Sort Layout 1 Right click on a Tube in an open Experiment and choose New Sort Layout Alternatively select a Tube in the Browser and click the Sort Layout button in the Workspace toolbar E 2 Inthe Sort Layout window choose the type of device from the Device menu Default sorting devices are listed along with any defined custom devices The Sort Layout window changes depending on the selected device the number of rows and columns in the window matches the number of tubes wells or spots in the collection device 3 Choose the Sort Precision mode from the Precision menu For more information see Sort Precision Modes on page 73 BD FACSAria User s Guide 4 Enter the number of events to be sorted in the Target Events field Once defined the number of events can be reused by choosing from the drop down menu For continuous sorting choose Continuous from the Target Events menu 5 Select the
158. ou need to adjust the PMT voltage for a subsequent compensation Tube you will need to record all compensation Tubes again 126 BD FACSAria User s Guide Calculating Compensation Before you can calculate compensation you will need to record data for each single stained control 1 2 Install the first stained control tube onto the cytometer In the Acquisition Controls frame click the Next button and then click Load Next moves the Acquisition pointer to the next Tube in the Browser Alternatively click to move the pointer to the next Tube Adjust the P1 gate to encircle the population of interest Ctrl click the Acquisition pointer to record data When recording is finished click Unload and install the next stained control tube onto the cytometer Repeat steps 2 through 5 until data for all stained control tubes has been recorded Now that data has been recorded you need to create regions around the fluorescence positive populations on the histogram for each Stained Control Tube 7 Double click the first Stained Control Tube FITC Stained Control to locate the corresponding plots on the worksheet Create an Interval gate around the fluorescence negative and positive populations on the histogram Figure 4 10 on page 128 Mi Tip Use the Auto Interval tool to quickly create an Interval gate around a population To keep the tool selected so you don t have to click the tool for each remaining Tube hold dow
159. path Click OK to stop running ethanol click Cancel to stop preparing for aseptic sort Sal Let the ethanol run as long as you like click OK when finished NOTICE Click Cancel only if you want to cancel the cleaning mode Do not click Cancel to stop running ethanol BD FACSAria User s Guide The instrument stops running the ethanol sample unloads the tube and stops the stream Replacing the Ethanol with Sheath Fluid Now that all fluid lines have been cleaned the system prompts you to install an autoclaved sheath container on the fluidic cart Follow these steps to complete the cleaning procedure NAseptic Sort G To finish preparing for an aseptic sort do the following 1 Replace the sheath tank with an autoclaved sheath container 2 Prime the system with sterile sheath 3 Replace the bypass tubing with a sterile filter 4 Bleed the air out ofthe sterile filter 5 Run Fluidics Startup fluidics shutdown fluidics startup 6 Load a tube with water and run it for 30 minutes 1 2 M 4 At the prompt detach the ethanol line from the Sheath port on the fluidics cart and connect it to the Ethanol port Figure 6 3 on page 180 Connect an autoclaved sheath container to the Sheath port Tip To prevent contamination autoclave the sheath probe and tubing along with the container Fill the container with sterile sheath fluid Prime the system with sterile sheath fluid e Click OK to dismiss
160. placement parts Item Part No Autoclavable 10 L container 333503 Auxiliary 5 L container 333504 Sheath sensor probe 5 level 334914 Waste sensor probe 6 level 334915 Auxiliary sensor probe 1 level 334911 Air filter for plenum cabinet side door 334351 Air filter for ACDU cabinet 334821 single 333595 set of 3 ULPA filter and tubing replacement kit set of 3 for use with the AMO 334822 Laser Specifications The following class 3B lasers are mounted on the BD FACSAria instrument Manufacturer Model Wavelength rowel nm mW Coherent Sapphire 488 20 488 13 2 3 JDS Uniphase 1144 P 633 11 wn Y 3 Point Source IFlex 2000 P 1 405 407 10 a 0 65 10 S StokerYale Lasiris SNF 701L 660 35 1 deg 660 35 3 3 single line laser ES 2 Appendix A Supplies and Options 215 Consumables Instrument Setup Particles 877 232 8995 Particle Supplier Catalog No BD CaliBRITE Beads BD Biosciences e Two color kit eee e 349502 unlabeled FITC PE e Three color kit e 340486 unlabeled FITC PE PerCP e PerCP beads e 340497 e PerCP Cy5 5 beads e 345036 e APC beads e 340487 Fluoresbrite Yellow Green 2 4m Polysciences Inc 18604 beads for the 407 nm laser 800 523 2575 SPHERO Rainbow Calibration BD Biosciences e 559123 Particles 3 0 3 4 um Pharmingen 8 peaks 877 232 8995 556286 e 556291 bright
161. pot monitors the breakoff and makes adjustments as necessary to maintain the breakoff throughout the day 142 BD FACSAria User s Guide xj Stream M Sweet Spot If the amplitude is lt 10 volts turn on Attenuation in the Side ical Stream Dal Sweet Spot o mpl 33 4 mpl 312 Freq 900 Freq 90 0 Drop 1 256 256 Drop 1 256 138 Ga 6 6 Ga 6 5 good satellite poor satellite formation formation Setting Up for Bulk Sorting This section describes how to set up the streams for two or four way sorting For sorting using the ACDU option see Setting Up for Sorting Into a Plate or Slide on page 153 AS WARNING Any instrument surface that comes in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling sorting hardware Wear suitable protective clothing and gloves 1 Install collection tubes in the required collection device Collection tube holders are available for 1 mL microtubes 12 x 75 mm tubes and 15 mL centrifuge tubes For compatible tubes see Labware on page 218 Install the tube holder on the instrument Close the sort block door and open the sort collection chamber door if needed The sort block door must be closed in order to open the collection chamber door Before installing the tube holder ensure that an O ring is installed in the groove on top of the tube holder The O ring minimizes the cha
162. r drawer is in WASTE position The drawer must be in SORT position for drops to reach output device Click OK to move drawer to SORT position and start sort Click Cancel to start sort with drawer in WASTE position Cancel There is no need to collect the beads when the drawer is closed the beads are sorted to waste 146 BD FACSAria User s Guide 6 9 Click the Optical Filter control in the Side Stream window This control moves the emission filter that allows you to view the Accudrop beads in front of the lower camera When the control is clicked the image switches from a raw image to a processed digitized image The two boxes indicate the region of the image where the left and center stream intensities are calculated during image processing the numbers shown are percentages of the total intensity If the left side stream is not completely contained in the left region adjust the voltage slider to place the stream in the center of the region Adjust the micrometer dial to obtain the brightest bead spot on the center stream Figure 1 17 on page 41 Optimize the drop delay Adjust the Drop Delay value in 1 drop increments Ctrl click arrow control to achieve close to 100 intensity in the left side stream Wait a few seconds after each click for a complete response to the Delay change Voltage Center Plate Voltage 4 000 2nd Drop 201 3rd Drop 10H 4th Drop oS Phase oal Figure 5 1 Sort
163. r not set to clicking Load or Acquire current Tube Recommended Solutions Click to move the Acquisition pointer in front of the appropriate Tube Laser shutter engaged Make sure the flow cell access door is completely closed Laser power off Turn on the laser power Viewing plots for a different Tube Double click the current Tube in the Browser to display the plots for that Tube Incorrect population s in plot Right click the plot and choose Show Populations Verify that the appropriate populations are displayed Chapter 7 Troubleshooting 203 Acquisition Troubleshooting continued Observation Possible Causes No events in plots after Uncolored events in plot clicking Acquire continued Recommended Solutions e Format the plot to display all events e Assign a color to the population displayed in the plot e Verify the population drawing order Current Instrument Configuration different from optical setup Verify that the instrument optics setup matches the current Instrument Configuration See Application Options on page 220 No sample in tube Add sample to tube or install new sample tube Sample not mixed properly Increase the Sample Agitation rate See Sample Agitation on page 81 Threshold not set to correct parameter usually FSC Set the threshold to the correct parameter for your application Multiple Threshold parameters not set corre
164. red device Device Setup Custom Device_001 Name Custom Device_001 Rows E Home Pos 0 0 Columns 1 Farthest Pos 0 0 Go to Home Set Home Go Farthest Set Farthest t e mu Y 158 BD FACSAria User s Guide Select the text in the Name field and enter a new name Enter the number of sort location Rows and Columns A device can have up to 60 rows and 25 columns Use the Arrow keys and the Test Sort button to set the Home location click Set Home See Setting Up the Stream on page 155 for details There are no default values for custom devices so greater initial adjustment with the Arrow keys is required Use the same procedure to set the Farthest location click Set Farthest The Farthest sort location is the well or spot on the lower right corner of the collection device Click Apply then Close After you set the Home and Farthest locations custom devices are listed in the Device drop down menu in the Sort Layout view NOTICE Once custom devices are defined you cannot change the number of rows and columns Click the Voltage control J to turn off the deflection plates Proceed with Determining the Drop Delay on page 145 and Sorting on page 149 Chapter 5 Sorting 159 Deleting a Custom Device 1 Choose Sort gt Custom Devices 2 Select the name of the custom device to be deleted in the Custom Devices dialog box 3 Click Delete The device is deleted from the Custom Dev
165. rochromes up to three lasers and up to 15 detectors Lit Instrument Configuration Current Configuration 3 laser 9 color Side Scatter Parameter Blue F zl Parameter Laser Detector FSC Blue FSC a ssc Blue F FITC Blue E PE Blue D PE Texas Red Blue Cc PerCP Cy5 5 Blue B PE Cy7 Blue JA APC Red B APC Cy7 Red JA Violett Violet B Violet2 Violet lA lx Add Configuration Delete Configuration Set Configuration Add Parameter Delete Parameter OK Cancel For the BD FACSAria instrument default configurations are provided for two and three laser systems You can switch between the default configurations depending on your system setup and application Examples of instrument configurations for common applications can be found in Application Options on page 220 In addition you can define custom configurations or add or modify the labels in an existing configuration See Defining a New Configuration on page 224 for instructions Selections made in the Instrument Configuration dialog box will determine which parameters are listed on the Parameters tab in the Inspector or the Instrument frame If you use one PMT detector to measure multiple fluorochromes you can list all possible fluorochromes in the Instrument Configuration dialog box and choose the appropriate fluorochrome in the Parameters tab A NOTICE For accurate data results the instrument optics setup must match the current Ins
166. rogation 30 line backflush 170 changing 184 pressure 48 temperature 82 102 sample optimization about 121 Experiment 122 samples running 131 satellites drop 142 Save Conflicts 93 saving analyses 135 sort conflicts 93 scaling area 112 113 119 scatter parameters distorted 207 Setup compensation 128 sheath flow 46 fluid 102 pressure 46 81 refilling 103 shutter laser 26 shutting down computer 164 fluidics 80 162 lasers 162 side streams optimizing 144 155 window 65 signals about 49 detection 51 fluorescent 50 generating 57 low Area 205 no fluorescent 205 scattered light 49 troubleshooting 208 Single Cell mode 73 slides installing 154 sorting into 153 sort block 32 collection chamber 35 Sort button 94 Sort Layouts about 68 90 creating 92 149 custom 88 158 editing 94 entering populations 93 Index 235 Sort Precision modes about 69 creating 75 88 defaults 73 deleting 75 Fine Tune 74 Four Way Purity 149 starting computer 100 fluidics 46 80 101 instrument 100 lasers 101 sorting 94 151 stream 101 Initial 74 Stokes shift 50 Purity 73 stop button 43 Single Cell 73 stopping sorting 94 Yield 73 stream Sort Reports centering 66 about 97 charging wire 140 exporting 98 control 63 printing 98 deflecting 66 Sort Setup values 89 flow rate 47 sorting starting 101 about 60 137 analysis 139 aseptic 178 aspirator drawer control 95 collection devices 91 conflicts 69 93 95 c
167. rs Indicator Level of Severity WARNING The hazard or unsafe practice could result in severe injury or death CAUTION The hazard or unsafe practice could result in minor injury NOTICE The hazard or unsafe practice could result in a possibly dangerous situation material could be damaged or data could be lost a In compliance with the ANSI Z535 4 standard Product Safety Signs and Labels x BD FACSAria User s Guide Table 3 Text and keyboard conventions Convention Use 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 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 7 Troubleshooting If additional assistance is required contact your local BD Biosciences technical support representative or supplier When contacting BD Biosciences have the following information available e product name part number and serial number e any error messages e
168. rument trame Communication failure between workstation and instrument e Quit the software and then restart it e If restarting does not work reset the instrument electronics switch off the main power wait 10 seconds until the system is fully depressurized and then switch the power back on e Restart the computer and the instrument Ethernet cable disconnected between workstation and instrument Unplug and then plug in the cable connectors and make sure they are secure IP address changed Enter the correct IP address Call BD Biosciences for assistance Master DAQ Overflow Event rate too high in Instrument frame Decrease the event rate or verify the threshold Instrument not Unknown responding in Status tab Perform the suggestions for a communication failure above 210 BD FACSAria User s Guide Appendix A Supplies and Options This appendix provides a list of supplies and options that are available for the BD FACSAria instrument e To order spare parts and consumables from BD Biosciences from within the US call 877 232 8995 or go to www bdbiosciences com In other countries contact your local BD Biosciences representative e To order instrument options contact your sales representative NOTICE This information is correct at the time of publication for up to date information refer to our website http www bdbiosciences com 211 Instrument Supplies
169. s gt Sample Line Backflush 2 Click Start to start the backflush N Sample Line Backflush C Click Start to start the backflush click Stop or Cancel when 2 the backflush is complete Start Stop Cancel 3 Click Stop to stop the backflush or click Cancel to stop the backflush and close the dialog box NOTICE The backflush does not stop automatically 170 BD FACSAria User s Guide Clean Flowcell Use the Clean Flowcell command to run a tube of cleaning fluid such as BD FACSRinse solution BD Catalog No 340346 through the sample line and flow cell without sheath running After the procedure is complete the cleaning fluid remains in the sample line and flow cell until the stream is restarted Note that the Clean Flowcell procedure is run automatically during fluidics shutdown You can repeat the procedure when poor optical performance indicates that additional cleaning is needed 1 2 Turn off the lasers and the stream if they are on Choose Instrument gt Cleaning Modes gt Clean Flowcell When prompted install a tube containing approximately 3 mL of cleaning fluid such as BD FACSRinse solution and click OK E Confirm 2 Install a tube with cleaning solution on the loading port and click OK Cancel The instrument loads the tube and cleans the cuvette with the cleaning solution A progress message appears during the cleaning process click OK to dismiss the message if you would like
170. s cart is not functioning properly If this occurs contact your BD Biosciences service representative for assistance Do not operate the instrument outside the normal air pressure range pressure gauge at approximately 88 psi Figure 1 5 Fluidics cart flow guage See the following sections for more information about the fluidics cart e Setting Up the Fluidics Cart on page 102 e Refilling Containers on page 103 e Emptying the Waste on page 104 Chapter 1 BD FACSAria Instrument Components 25 e Scheduled Maintenance on page 168 e Fluidics Cart Troubleshooting on page 209 Flow Cytometer The benchtop flow cytometer contains the major components for all three subsystems fluidics optics and electronics The BD FACSAria instrument is relatively compact with a much smaller footprint than most sorters with the same capabilities The instrument can be set up on a typical laboratory benchtop or table and it requires only a standard electrical outlet No special facilities are required flow cell access door power panel optics access door sort collection chamber da gt side door sample injection chamber Figure 1 6 BD FACSAria flow cytometer To view the fluidics components open the side door and lift the flow cell access door to view the optics components open the optics access door The power panel and connectors are found on the left side of the instrument Other electronic components are embedded within the
171. se trails off Signal Intensity gt Time gt Figure 2 12 Anatomy of a pulse Chapter 2 Theory of Operation 57 58 Pulse Parameters A parameter is a pulse property that is generated by a single photomultiplier tube or photodiode measuring fluorescent or scattered light You can measure three characteristics of a pulse the area height or width The pulse height measures the maximum digitized value for the pulse the pulse area calculates the sum of all height areas for the pulse and the pulse width calculates 21 2_ x 64000 height You can select which pulse characteristics you want to measure for each parameter using the signal intensity gt pulse height time gt Parameters tab of the Tube Inspector Pulse area A is measured by default The Parameters tab also contains Voltage controls that allow you to amplify signals by applying a voltage to PMTs or an electronic gain to FSC signal As the voltage is increased the detector sensitivity increases resulting in increased signal As the voltage is decreased the detector sensitivity decreases resulting in decreased signal Tube Labels Acq Instr Settings Keywords Parameters Threshold Compensation Ratio Parameter Voltage Log La H w r FSC 260 DB DE r 55C 300 piminio FITC 500 OMIM p PE 500 D M M O r PE Texas Red 500 DIM M b PerCP Cy5 5 500 DIPHIB IO PE Cy7 50
172. se until the light turns off and the button returns to its original position NOTICE The Emergency stop button does not turn off the lasers or shut down the instrument main power Chapter 1 BD FACSAria Instrument Components 43 BD FACSAria Workstation 44 Data acquisition and analysis as well as most BD FACSAria instrument functions are controlled by BD FACSDiVa software on the BD FACSAria workstation The workstation includes a desktop computer one or two monitors and a color printer and is equipped with the following applications e Microsoft Windows 2000 operating system e BD FACSDiVa software Data acquisition and analysis Automatic fluidics startup shutdown and cleaning modes Sort setup stream monitoring clog detection and recovery e Supporting documentation for the software For information about using BD FACSAria specific software features read Chapter 2 Theory of Operation and Chapter 3 Using BD FACSDiVa Software For more information about general software features refer to the BD FACSDiVa Software User s Guide Mi Tip Information about the software can be easily accessed by choosing User s Guide from the BD FACSDiVa Help menu BD FACSAria User s Guide 2 Theory of Operation This chapter describes how the BD FACSAria instrument works and how BD FACSDiVa software components are used to operate different system components For a general overview of the software see Chapter 3 See the followi
173. settings icon and choose Print Chapter 4 Running Samples 131 Analyzing Data This section describes how to set up plots gates and a statistics view to analyze the recorded data By the end of this section your analysis should look similar to that shown in Figure 4 11 on page 134 1 Create a new Acquisition Template rename the template T B NK Analysis 2 Select the first Tube under the LWB Specimen and create the following plots on the Analysis template e FSC vs SSC e CD3 PerCP Cy5 5 vs CD16 56 PE e CD3 PerCP Cy5 5 vs CD19 APC e CD3 PerCP Cy5 5 vs CD8 APC Cy7 e CD3 PerCP CyS5 5 vs CD4 FITC 3 Resize the plots so they fit on one page as shown in Figure 4 11 on page 134 4 Draw a gate around the lymphocytes use the Population Hierarchy to rename the population Lymphocytes 5 Select all plots except the FSC vs SSC plot and specify to show only the Lymphocyte population 6 Select all plots and click the Title tab in the Plot Inspector select the checkboxes to display the Tube and Population names in the plot titles Inspector ol x Plot Title Labels Histogram Title Content Y Tube Populations 132 BD FACSAria User s Guide 7 Create a Statistics view edit the view to show only the Lymphocyte population and to display the mean for all fluorochromes 8 Draw a region around the CD3 positive population on the CD3 PerCP CyS 5 vs CD16 56 PE plot name the population T Cells Vv Tip When drawing regions be
174. so it would have to be replaced again 8 Check the fitting connections to make sure they are not leaking Turn on the stream load a tube of water and make sure none of the fittings are leaking If needed unload the tube turn off the stream and tighten the fittings 186 BD FACSAria User s Guide AS WARNING Make sure all compression fittings are securely tightened If any 9 fitting is loose the tubing could detach during high pressure operation exposing the operator to potentially biohazardous sample spray Verify the length of the sample line The sample line should not bow or bend when a tube is loaded If you need to adjust the length unscrew the nut on top of the sample injection chamber adjust the length and tighten the nut again Changing Air Filters The BD FACSAria instrument has two air filters one in the sort collection chamber door and one in the side door To change the filter in the sort collection chamber door slide out the old filter and slide in the new filter Refer to the documentation provided with the Aerosol Management Option for more information To change the air filter in the side door turn the pegs at each corner of the filter and remove the old filter Install a new filter and turn the pegs to hold the filter in place Replace the filter annually to semi annually depending on the quality of the air in the laboratory Chapter 6 Shutdown and Maintenance 187 Unscheduled Maintenance T
175. so the same configuration can be used for all Experiments Changing instrument configurations within the same Experiment can generate errors Mi Tip To ensure that an Experiment contains the correct parameters set the configuration you want to use before creating a new Experiment 224 BD FACSAria User s Guide 1 Choose Instrument gt Instrument Configuration The Instrument Configuration dialog box appears Lit instrument Configuration Current Configuration 1 laser 3 color E ue F E Parameter Laser Detector FSC Blue FSC ssc Blue F FITC Blue E PE Blue o PE Texas Red Blue c PerCP Cy5 5 Blue B PE Cy7 Blue lA APC Red B JAPC Cy7 Red A Add Configuration Delete Configuration Set Configuration Add Parameter Dee Parameter OK Cancel 2 Click the Add Configuration button Alternatively you can use the Add Parameter button to add parameters to an existing configuration You can define multiple parameters for the same Laser Detector position and within each Experiment use the Parameter tab in the Tube Inspector to specify which parameter to detect Only one parameter can be detected from each PMT per Experiment NOTICE Instrument configurations that have a compensation Setup associated with them cannot be edited To edit an existing configuration all Setups associated with the configuration must be deleted from the Setup catalog NOTICE Use the Experiment Layout dialog box to label Tubes wit
176. starting 100 status report 85 supplies 212 workstation 44 Instrument frame 79 interrogation point 30 L labels parameter 130 precaution xvii label specific Tubes 123 lasers about 36 adjusting delay 118 delay 59 diode 41 67 optimizing signal 112 power 42 safety xiii shutter 26 shutting down 162 specifications 215 starting 101 warmup time 101 Layout Sort See Sort Layouts levels fluid 83 100 sample 29 lever nozzle 140 166 light detection 51 injection chamber 29 scatter signals 49 voltage warning 33 Load button 86 loading tubes 29 47 longpass filters 53 M maintenance 168 188 Masks about 69 default Precision Modes 73 Phase 72 Purity 71 Yield 69 Master DAQ overflow error 210 micrometer dial 41 147 mirrors dichroic 53 modes defining 75 deleting 75 Sort Precision 69 73 monitoring sorts 96 152 N Next button 107 nozzles about 31 changing 140 cleaning 188 lever 140 166 O ring 140 recommended sizes 89 140 O octagon about 38 39 51 default filters 40 220 221 opening aspirator drawer 95 151 sample injection chamber 29 Optical Filter control 66 67 optical filters about 52 changing 223 cleaning 192 default setup 40 220 221 optics collection 38 default setup 40 excitation 36 fiber 37 instrument 36 reordering 212 stream viewing 40 Index 233 optimization instrument 105 sample 121 ordering supplies 211 O ring nozzle 140 P param
177. stream and fine tune the side streams if needed Generally the Sort Setup mode provides good starting values for these settings Adjust the values only if needed to optimize the streams 6 Open the aspirator drawer and aim the side stream s into the bottom of each collection tube Open an Experiment if one is not already open and create a Sort Layout for any of the Tubes e In the Sort Layout view click the Aspirator Drawer button to open the drawer 24 e Open the sort block door and aim each side stream into the bottom of a tube as you adjust the corresponding slider control e When you are satisfied with the side stream deflection close the sort block door 144 BD FACSAria User s Guide A WARNING A 12 000 volt potential exists between the deflection plates when they are on Contact with the charged plates results in serious electrical shock Do not touch the deflection plates when the plate voltage is on The plates remain energized even when the sort block door is opened 7 Click the Voltage control J to turn off the deflection plates Determining the Drop Delay BD FACS Accudrop technology is used to determine the optimal drop delay setting for your sorting application For more information see Setting the Drop Delay on page 67 Before beginning this procedure make sure the stream is stable and the Sweet Spot is on Setting Up the Experiment The steps in this section show you how to set up an Experim
178. sure to include events on the plot axes 9 Draw a region around the CD16 56 positive population on the same plot name the population NK Cells 10 Draw a region around the CD19 population on the CD3 PerCP Cy5 5 vs CD19 APC plot name the population B Cells 11 Select the T cell population in the Population Hierarchy view draw a region around the double positive population on the CD3 PerCP Cy5 5 vs CD8 APC Cy7 plot and name the population T Cytotoxic Because the T cell population is selected first the T cytotoxic cells become a subset of all T cells 12 Select the T cell population in the Population Hierarchy view draw a region around the double positive population on the CD3 PerCP CyS 5 vs CD4 FITC plot and name the population T Helper 13 Print the analysis See Figure 4 11 on page 134 for an example Chapter 4 Running Samples 133 e 8 3 a 2 o a a 50 100 150 200 250 FSC A 1 000 Dal Population Tube T B NK_001 Events Ml Lymphocytes Wit cells DIT cytotoxic HT Helper W nk Cells O B Cells 21 0 69 7 22 7 73 3 12 0 8 9 Events Parent Total 100 0 21 0 14 6 3 3 10 7 25 1 9 TIBINK_001 Lymphocytes 10 10 10 CDA FITC A 10 10 10 10 10 CD3 PerCP Cy5 5 A S 104 10 10 CD16 56 PE A 10 T BINK_001 Lymphocytes 10 10 10 10 CD3 PerCP Cy5 5 A S TIBINK_001 Lymphocytes TA CD19 APC A
179. t Components 37 Collection Optics From the cuvette flow cell laser light is collected by a fluorescent objective lens that is gel coupled to the cuvette to transmit the maximum amount of light The lens collects and focuses fluorescent light emitted at each of the laser focal points onto individual collection fibers These fibers transfer the emitted light to the collection optics as shown in the following figure individual collection fibers E M fluorescent objective lens cuvette Y stream The collection optics are set up in octagon and trigon shaped arrays that are engineered to maximize signal detection from each laser This is accomplished by transmitting the highest wavelengths to the first photomultiplier tube PMT and reflecting lower wavelengths to the next PMT through a series of long pass dichroic mirrors Bandpass filters in front of each PMT allow fine tuning of the spectral wavelengths that need to be collected Since reflection is more efficient than transmittance this design greatly increases the multicolor detection capabilities of the instrument 38 BD FACSAria User s Guide Detectors A standard system is equipped with an octagon containing six PMTs which detect light from the 488 nm blue laser and a trigon containing two PMTs which detect light from the 633 nm red laser The third laser option adds an additional trigon containing two PMTs to detect light from the 407 nm violet
180. tains samples stained with the same fluorophore conjugated to different antibodies labels that require different compensation values Chapter 4 Running Samples 123 This is especially noticeable in tandem conjugates due to lot to lot variation Refer to the BD FACSDiVa Software User s Guide for more information about this feature Adjusting the Voltages and Threshold The unstained control will be used to check for nonspecific antibody binding to adjust forward scatter side scatter and FSC threshold to gate the population of interest lymphocytes in this case and to adjust fluorescence settings 1 Install the unstained control tube on the cytometer 2 Verify that the green Acquisition pointer is in front of the Unstained Control Tube in the Browser click Load 3 Adjust the FSC and SSC voltages to appropriately display the scatter properties of the LWB sample Figure 4 7 Unstained Control oe 3 3a a 38 we Ze SF ao oe 50 100 1 50 200 FSC A 250 1 000 Figure 4 7 Voltages adjusted 4 Click the Threshold tab and adjust the FSC Threshold if needed Set the threshold to remove most of the debris without cutting off the lymphocyte population Figure 4 7 124 BD FACSAria User s Guide 5 Adjust the P1 gate on the Unstained Control worksheet to surround only the lymphocyte population Figure 4 7 Select the gate by clicking on the boundary Once selected you can drag the gate to move it or dra
181. teria but were not sorted because of conflicts e Conflict Rate number of conflicts second e Efficiency number of sorted events sort conflicts sorted events x 100 Monitoring a Sort During sorting each sort location field displays the number of actual sorted events When a target number is specified the field displays the actual number of events along with the number of target events Figure 3 8 A progress bar appears behind the Sort Rate counter field showing the progress of the sort 2 35 psi_001 4way rectangle x Device Precision Target Events Save Conflicts ls Tube y frorsz x y D Far Left Right Far Right Brz 251744 P3 264697 P4 268980 P5 259199 Sort Rate 960 evtis 1010 evtis 1026 evtis 989 evtis Conf Cnt 101696 evt 107034evt 109057 evt 104484 evt Confl Rate 388 evtis 408 evtis 416 evtis 398 evtis _Efficiency 71 71 71 71 Sort wras aj View Counters Figure 3 8 Sort Layout during sorting BD FACSAria User s Guide Sort Report Choose Sort gt Sort Report to view a report of the current Sort Layout This menu item is enabled only if a Sort Layout is open and the instrument is not sorting The Sort Report can be printed or exported Mi Tip To save counter information print a Sort Report immediately after sorting When you close the Sort Layout all counter information is lost A Sort Report contains the following e Header informatio
182. th Pressure Clean Flowcell Sample Agitation gt Prime after Tank Refill Sample Temperature Long Clean Instrument Configuration Prepare for Aseptic Sort BD FACSAria User s Guide Sheath Pressure The sheath pressure determines how quickly particles pass through the laser beam Choose Instrument gt Sheath Pressure to access a dialog box where you can view the current sheath pressure i Sheath Pressure ox Sheath Pressure 70 00 JH A NOTICE Fach Sort Setup option is optimized at a preset sheath pressure If you change the sheath pressure a multitude of other values will be affected including the drop drive frequency drop charge levels laser delay area scaling factor and other values For proper instrument operation change the sheath pressure by choosing an option from the Sort gt Sort Setup menu do not adjust the pressure using controls in the Sheath Pressure dialog box Note that the Sheath Pressure command is disabled when the Sweet Spot is on Sample Agitation Choose Instrument gt Sample Agitation to specify the speed at which samples are agitated You can choose from one of the specified values or choose Off to turn off agitation Fluidics Startup Fluidics Shutdown Cleaning Modes Sheath Pressure Sample Agitation Off Sample Temperature 300rpm Instrument Configuration 200rpm Instrument Name e 100rpm Chapter 3 Using BD FACSDiVa Software 81 82
183. the instruction message and choose Instrument gt Cleaning Modes gt Prime After Tank Refill e Select the Sheath checkbox click OK When the prime is finished remove the bypass tubing and install a new sheath filter in its place Open the bleeder valve on top of the filter and wait for fluid to seep out of the bleeder valve close the valve See Purging Filters on page 182 Chapter 6 Shutdown and Maintenance 181 6 Choose Instrument gt Fluidics Startup For additional decontamination run the Fluidics Shutdown procedure when the system is ready and then repeat the Fluidics Startup procedure again 7 Install a tube of DI water on the loading port load the tube and run it for 10 minutes 8 Optional Run the stream for an additional 10 20 minutes Purging Filters Once a week purge air from all fluid filters by opening the bleeder valve on the top of each filter in turn This ensures that the filters will not dry out 1 Unscrew the bleeder valve on the top of the filter 2 Wait until fluid seeps out through the valve 3 Close the valve 182 BD FACSAria User s Guide Changing Fluid Filters BD Biosciences recommends changing the fluid filters every 3 6 months or when increased debris in an FSC vs SSC plot indicate that the sheath filter needs to be replaced Spare filters are included with the accessory kit filter 1 Remove the filter by pressing the tabs on each quick disconnect coupling 2 Install
184. the nozzle grounding plate lever spring and plunger along with the hole in the end of the lever the lever well and the nozzle well nozzle well lever well Thoroughly dry the disassembled parts and the exposed instrument surfaces with a Kimwipe or a cotton swab NOTICE If you use a swab make sure no cotton fibers are left behind Reassemble and reinstall the nozzle locking lever Insert the small end of the plunger and then the spring into the lever hole as shown in Figure 6 2 Install the lever at an angle to compress the spring against the side of the lever well Once the spring is fully compressed straighten out the lever and push it all the way to the back Turn the lever 180 to the left to lock the lever in place Reinstall the grounding plate and the nozzle NOTICE Install the grounding plate with the notched end toward you notched Make sure an O ring is installed inside the groove at end the end of the nozzle Insert the nozzle at a slight Chapter 6 Shutdown and Maintenance 167 downward angle to avoid contact between the O ring and the cuvette Push it gently all the way forward without rocking it from side to side 11 Turn the nozzle locking lever clockwise to the 12 00 position and plug the stream charging wire into the nozzle 12 Restart the stream and verify it runs smoothly through the grounding plate If you need further assistance see Troubleshooting the S
185. tial hazard Do not remove these labels Use appropriate precaution to avoid injury by the indicated hazard See the previous sections in this booklet for more information Label Location s Potential Hazard Waste tank waste tank Risk of exposure to connectors on fluidics cart biologically transmissible disease Safety and Limitations xvii Label Location s Potential Hazard Waste tank cap Risk of exposure to biologically transmissible disease Ethanol tank ethanol tank connectors on fluidics cart Highly flammable material Ethanol tank cap Highly flammable material On or near all removable covers and any place where the laser beam can emerge from the instrument Risk of exposure to hazardous laser radiation BD FACSAria User s Guide Label Location s Potential Hazard A Deflection Plates Sort block door and deflection plates warning light Dangerous voltage Behind sample injection chamber Risk of crushing or pinching by moving parts CAUTION MOVING PARTS Floor of sort collection chamber Risk of crushing or pinching by moving parts A WARNING Before applying power configure the power input module to the supply voltage Consult the user s guide for instructions Peel Here Power input on fluidics cart Risk of instrument damage if wrong voltage applied a NOTE Voltage is set by your service
186. tream on page 194 Scheduled Maintenance For optimal instrument functioning perform the following procedures according to the recommended maintenance schedule Procedure Recommended Frequency Internal Cleaning on page 168 See Table 6 1 on page 169 Purging Filters on page 182 Weekly Changing Fluid Filters on page 183 Every 3 6 months Changing the Sample Lines on page 184 Every 4 6 months or when required Changing Air Filters on page 187 Every 6 12 months depending on instrument use and the quality of the air Internal Cleaning BD FACSDiVa software includes four pre programmed cleaning modes that can be used singly or in combination to provide the required level of cleaning The following sections describe what actions are taken when you choose the indicated command from the Instrument gt Cleaning Modes menu See Table 6 1 on page 169 for an overview of each mode 168 BD FACSAria User s Guide Fluidics Startup Fluidics Shutdown Cleaning Modes Sample Line Backflush Sheath Pressure Clean Flowcell Sample Agitation gt Prime after Tank Refill Sample Temperature Long Clean Instrument Configuration Prepare for Aseptic Sort Table 6 1 Instrument cleaning modes Cleaning Mode Summary Frequency Sample Line Backflush on Flushes the sample line with After running samples page 170 sheath fluid with adherent cells or dye Clean Flowcell on Cleans the sample path and When in
187. tream window The voltage warning light illuminates indicating that the plates are charged NOTICE Make sure the center stream image does not move after the plates are turned on If the stream image is off center use the Voltage Center slider to re center the stream Major movement of the center stream could indicate that the plates or area around the plates needs cleaning See External Cleaning on page 164 3 Turn on Test Sort and optimize the far left side stream turn off Test Sort Adjust the far left slider for minimal deflection of the stream The stream should be deflected just enough to clear the hole in the splash shield Do not adjust the other sliders Tf you cannot see a stream image or the image is dim adjust the micrometer dial on the diode laser to better view the streams Figure 1 17 on page 41 4 Choose Sort gt Home Device 5 In the Device Setup dialog box choose the collection device you are using and click Go to Home Device Setup 6 Well Falcon Name 96 Well Falcon 24 Well Falcon Home Pos 855 7700 acl Falcon Go to Home Set Home 96 Well Falcon 384 Well Falcon Slide Frosted End Slide Standard Test Sort button t EME Apply Close The stage moves to the preprogrammed Home position 156 BD FACSAria User s Guide 10 11 12 13 Double click the Test Sort button to deposit a drop at the Home location Inspect the collection device to see where t
188. trol on and then off Sonicate the nozzle and reinstall it 19 BD FACSAria User s Guide Troubleshooting the Stream continued Observation Possible Causes Leaking or spraying Nozzle inserted improperly around nozzle Recommended Solutions Turn off the stream Remove the nozzle and the grounding plate and thoroughly clean and dry the plate and the area around the plate See Cleaning the Nozzle and Grounding Plate on page 165 for instructions Extra O ring blocking nozzle Remove the nozzle and use a cotton swab to clear out the cuvette Grounding plate inserted improperly Make sure the grounding plate is inserted with the notched end toward you See Cleaning the Nozzle and Grounding Plate on page 165 Drop breakoff too long Bubbles in flow cell Open the flow cell access door and check for bubbles in the flow cell If they are visible turn off the stream wait a few seconds and turn on the stream again Attenuation on Turn off attenuation Amplitude too low Increase the amplitude until you can see drops If you need a very high amplitude gt 70 volts to see drops you might have air bubbles in the flow cell Nozzle inserted improperly Turn off the stream Remove the nozzle and ensure that the O ring is in place Reinsert the nozzle at a slight downward angle to prevent loss or movement of the O ring Make sure the nozzle is completely seated against the back wall of the
189. trument Configuration BD FACSAria User s Guide Instrument Status Report Choose Instrument gt Instrument Status Report to view a report of the current instrument settings The Instrument Status Report is displayed in a separate window with a menu bar above the report header For a full description of the Instrument Status Report refer to the BD FACSDiVa Software User s Guide A BD FACSAria instrument report includes the following additional information e The Instrument Info section lists the sheath pressure and flow rate along with the lasers used corresponding Laser Delay and Area Scaling factors and the Window Extension e The Sort Settings section lists all sort setup values along with the Plate Voltage and Voltage Center values If the Sweet Spot is off Breakoff and Gap values are shown if the Sweet Spot is on Drop 1 and target Gap values are shown File Instrument FACSAria Instrument Status Report Date 2003 03 04 at 12 20 35 erial Number 1 Instrument Info Laser Delay Area Scaling Blue 0 0 1 0 Red 36 0 1 0 Violet 18 1 1 0 Window Extension 0 0 Sheath Pressure 40 00 Sample Flow Rate 1 0 Sort Settings Sort Setup High Frequency 90 00 Amplitude 80 00 Phase 0 00 Drop Delay 44 00 Attenuation Off Sweet Spot Off Breakoff 230 Gap 6 Plates Voltage 3 400 00 Voltage Centering 100 Chapter 3 Using BD FACSDiVa Software 85 86 Acquisition Controls Along with the controls described in th
190. uidics Startup procedure removes DI water from the plenum tanks and fluid lines and replaces it with sheath fluid When the Fluidics Startup procedure is complete use software controls to start the stream e Click the Sorting button in the Workspace toolbar Kaj to display the Breakoff and Side Stream windows e Click the Stream button in the Breakoff window to turn high x on the stream Stream B Sweet Spot Check the stream position in the waste aspirator and inspect the stream image in the Breakoff window The stream should flow smoothly from the nozzle into the center of the waste aspirator Drops should appear uniform and evenly spaced If the stream is flowing but it is unsteady open the flow cell access door and check for bubbles in the flow cell If you see bubbles turn off the stream wait a few seconds and turn on the stream again If you see any dripping or spraying or the stream image appears abnormal turn off the stream and go to Troubleshooting the Stream on page 194 Turn on the lasers wait 30 minutes for the lasers to warm up To turn on a laser press the corresponding laser power button in the power panel See Figure 4 1 on page 100 Chapter 4 Running Samples 101 Mi Tip If you are using temperature control start heating or cooling the sample injection chamber or the recirculating water bath while the lasers are warming up See Sample Temperature on page 82 8 Choose Sort gt Sort Setup and verif
191. vel descends below 20 of the tank capacity When this occurs the corresponding level indicator changes to black bleach ethanol sheath waste DI empty full Instrument Connected m i A Figure 3 3 Levels indicators When the sheath is low or the waste is full while the stream is running the corresponding indicator turns red and the following warning message is displayed every 5 minutes until the stream is turned off If the warning message is not dismissed the system automatically turns off the stream after 15 minutes Y Warning 2 Waste is full please empty Close A NOTICE Do not dismiss the warning message without refilling the sheath or emptying the waste If you continue to run the instrument after dismissing the message the waste container could overflow or the sheath container could run dry For a description of other controls in the Instrument frame refer to the BD FACSDiVa Software User s Guide Delay information specific to the BD FACSAria instrument is described in Laser Delay on page 59 Chapter 3 Using BD FACSDiVa Software 83 84 Instrument Configuration The BD FACSAria instrument is equipped with a specific set of lasers filters and dichroic mirrors The Instrument Configuration dialog box lets you define which fluorochromes or cell parameters will be measured at each photomultiplier tube PMT detector Within this dialog box you can define parameters for an unlimited number of fluo
192. x A aborts electronic 208 See also conflicts sort Access Stage button 95 accessory kit 213 Accudrop about 67 Experiment 145 optimizing drop delay 146 ACDU about 35 accessing stage 95 chamber 35 collection devices 156 custom devices 158 installing splash shield 153 sorting 153 troubleshooting 200 Acquire button 86 acquisition controls 79 86 events torecord 130 status frame 79 troubleshooting 203 Acquisition Templates adding Sort Layouts 92 creating 108 129 for instrument QC 108 previewing data 129 135 printing 116 adding Experiments 106 folders 106 instrument configurations 224 parameters 225 Sort Layouts 92 149 sort populations 93 statistics view 109 adjusting amplitude 64 141 area scaling 112 113 119 Drop 1 64 142 drop delay 146 flow rate 48 86 Home location 155 laser delay 118 micrometer dial 147 PMT voltages 58 124 side streams 144 155 sort block angle 32 141 threshold 124 window extension 118 aerosol containment 34 management option See AMO agitating samples 81 external 24 filters changing 187 pressure 22 25 227 AMO about 34 changing filters 187 connecting 23 amplitude about 63 adjusting 64 141 analysis data 115 129 immunophenotyping 132 reusing 135 saving 135 sorting 139 applications custom 222 recommended flow rates 49 recommended Sort Setup 89 area parameters 58 area scaling 112 113 119 aseptic sorting 178 aspirator drawer about
193. y the setup mode or choose a new mode For information about Sort Setup modes see Sort Setup on page 89 Setting Up the Fluidics Cart Mi Tip Check the fluid levels in the sheath and waste containers every time you use the instrument This ensures that you will not run out of sheath fluid during an experiment and that the waste container will not overflow Fluidics level indicators are shown in the Instrument frame in BD FACSDiVa software sheath waste DI bleach ethanol Instrument Connected E A T T If a fluidics container is running low refill it with the fluid indicated on the container label For best results fill the containers only with the fluids shown in Table 4 1 For ordering information see Reagents on page 217 For instructions on emptying the waste see page 104 Table 4 1 Recommended fluids Container Compatible Fluid Capacity Sheath e BD FACSFlow solution non sterile or Two 10 L containers or one e 1X phosphate buffered saline PBS with or without 20 L cubitainer preservatives Bleach e 10 household bleach 0 5 sodium hypochlorite or SL e BD FACSClean solution DI Deionized water SL Ethanol 70 solution diluted in DI or laboratory grade water SL 102 BD FACSAria User s Guide Refilling Containers You can refill a container directly on the fluidics cart without detaching any lines or you can detach the container for refilling Note that during operation you can add fluid to a conta
194. ylene 125 bag e 352096 e Polypropylene 50 rack e 352097 e Polystyrene 125 bag e 352095 e Polystyrene 50 rack e 352099 218 BD FACSAria User s Guide Instrument Options Your BD FACSAria instrument can be upgraded with the following options Contact your sales representative for more information Third laser option The third laser option upgrades your instrument with a violet laser a trigon and two PMTs for greater flexibility in analysis and sorting Optical upgrade Optical upgrades are available for two and three laser systems to maximize the detection capabilities of the instrument Each upgrade includes detectors and electronics to detect three or four additional colors respectively Aerosol Management Option AMO The AMO helps contain aerosols by evacuating the sort collection chamber during sorting The option includes a vacuum source attached to an ultra low penetrating air ULPA filter Temperature control option This option can be used to control the temperature of sorted samples using a recirculating water bath and metal collection tube holders Automated cell deposition unit ACDU The ACDU option provides hardware that allows you to sort into multiwell plates and onto slides Appendix A Supplies and Options 219 Application Options The octagon and trigon detector arrays in the BD FACSAria instrument allow many different combinations of mirrors a

BD FACSAria User's Guide

Contents

Download Pdf Manuals

Related Search

Related Contents