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BD FACSAria II User's Guide

1. NA BD FACSAria II User s Guide After you add a population the population and the number of target events are added to the corresponding sort location field Vv Tip Select a row or column header to select all fields in that row or column When you add a population it will be added to all selected fields at once 7 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 Fora two tube layout conflicts are sorted to the right amp Tube_001 Sort Layout_001 Device Precision Target Events Save Sort Reports Save Conflicts i r 2 Tube v Purity y lv Ask User v Left Right Left Conflicts Continuous e For a four tube layout conflicts for the far left tube are sorted to the left and conflicts for the far right tube are sorted to the right amp Tube_001 Sort Layout_001 Bl Device Precision Target Events Save Sort Reports Save Conflicts v purity v wv Ask User vi v Far Left i Left Right Far Right Left Conflicts Continu Right Conflicts Contin Editing a Sort Layout e To change the number of events for any population click in the Sort Location field s containing the population then select a number from the Target Events menu or enter a new number e To remove a population fr
2. Set up the fluorescence plots to display data from the FSC gate The FSC gate defines singlet events By gating the fluorescence plots through this population only singlet events will be recorded a Select the two plots b In the Inspector select the FSC Gate checkbox Arrange the fluorescence plots so they fill the page vertically For an example see Figure 4 27 on page 145 Chapter 4 Running Samples 141 Recording Data This section describes how to adjust the gates to eliminate doublets and record singlet events 1 Move the current tube pointer to the 4 color_001 tube 2 Install the first mixed sample tube onto the loading port and click Load 3 Change the Events to Display to 5 000 events 4 Adjust the Scatter Gate to encompass the singlet events 5 Adjust the SSC Gate to encompass the low SSC W population 6 Adjust the FSC Gate to encompass the low FSC W population Figure 4 26 Discriminating singlet events 4 color_001 All Events 4 color_001 FSC Gate 4 color_001 FSC Gate 190 150 200 250 a a a a a a 100 150 200 250 FSC WW amp 1 000 amp 3 x oO a a SSC A amp 1 000 SSC Gate FSC Gate _ Scatter Gate 60 100 150 200 250 50 100 150 200 250 50 100 160 200 250 FSc A 1 000 S8C H 1 000 FSC H amp 1 000 Tube 4 color_001 Population Events Parent Total W All Events 10 000 H 100 0 E Scatter Gate 9 341 93 4 E ssc Gate 9 337 100 0 H FSc Gate
3. OOOOOOO00000 OOOOOO00000 OOOOOOO00000 Quick disconnect Bleeder valve couplings 1 Remove the filter by pressing the tabs on each quick disconnect coupling See Figure 6 9 2 Install the new filter and connect the quick disconnect couplings Write the current date on the filter so you will know when to replace it Chapter 6 Shutdown and Maintenance 191 3 Open the bleeder valve on top of the filter a small amount and leave it open until fluid seeps out through the valve 4 Close the valve 5 Wipe up any excess fluid that might have dripped onto the fluidics cart Changing the Sheath Filter We recommend changing the sheath filter every 3 months or when increased debris in an FSC vs SSC plot indicates that the sheath filter needs to be replaced Spare filters are included with the accessory kit NOTE The sheath filter is the same as the one used for the ethanol shutdown filter The filters can be used for either purpose 1 Turn off the stream 2 Remove the filter by pressing the tabs on each quick disconnect coupling See Figure 6 10 Figure 6 10 Sheath filter Direction of flow gt 3 Unscrew the nuts at each end of the filter to remove the connecting tubing 4 Locate a new filter and wrap the threads at each end with Teflon tape 192 BD FACSAria II User s Guide Observe the direction of flow on the filter and replace it in the same orientation a
4. Load a tube with beads and cick Run button to start setup Characterize Chack Performance o Cytometer Configuration 130 20 Violet PerCP Cy5 5 AmC Select Configuration Setup Beads Lot ID 85631 s Produact CST Setup Beads Part 910723 Expeation Date 06 30 2009 Parameter Value Shutdown Soktion OK Cleaning Sokition OK Float Swatch OK Pump OK Waste Tank ox Sheath Pressure 9 87 Sheath Level 75 Sample Pressure 8 71 Connected Chapter 4 Running Samples 117 2 Verify that the bead lot information under Setup Beads matches the Cytometer Setup and Tracking bead lot Setup Beads 68341 v Product CST Setup Beads Part 683391 Expiration Date 10 10 2007 Select the correct lot ID from the menu The bead lot ID number is located on the CS amp T beads vial 3 Verify that the cytometer configuration is correct for your experiment Cytometer Configuration 3 laser 9 color 5 2 2 Configuration 7 BD default Lot ID 68341 If the cytometer is not set to the correct configuration a Click Select Configuration in the Setup Control window Setup Control RUO Load a tube with beads and click Run button to start setup Characterize Check Performance vv Gen Cytometer Configuration 4 Blue 2 Violet 2 355UV 2 Red Setup Beads Lot ID 68341 v Product CST Setup Beads Part 683391 10 10 2007 Expiration Date b Select the correct
5. Make adjustments to the experiment as needed Click the Resume button in the Sort Layout window to continue sorting Chapter 5 Sorting 167 Setting Up for Sorting Into a Plate or Slide The following sections describe how to set up for sorting into a plate or slide For general guidelines see Setting Up for Sorting on page 150 Installing the Sorting Hardware A Any cytometer 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 a 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 b Remove the tube holder if one is installed c Slide the splash shield into the slotted fittings below the sort aspirator drawer and push it all the way in 168 BD FACSAria II User s Guide Click the Access Stage button to bring the ACDU stage to the front a Open an experiment if one is not already open and create a sort layout for any of the tubes b In the Sort Layout window click the Access Stage button to move the stage to the front of the sort collection chamber Install the required collection device on the stage Figure 5 6 e If you are sorting into a plate install the plate with well A1 toward
6. x ny 2 yyy W wy Vy we k y INCHES W C x 2 o gt v INCHES W F FILTER FLOW GAUGE FILTER FLOW GAUGE New filter Used filter Setting Up for Sorting To get set up for sorting 1 Start up the flow cytometer system See Cytometer Startup on page 100 2 Start up the evacuator as described in Starting Up the Evacuator on page 278 3 Follow the standard sort setup procedure See Setting Up for Sorting on page 150 NOTE Always start up the evacuator before setting up for sorting If you start up the evacuator after sort setup is complete you will need to repeat the setup procedure Appendix C BD Aerosol Management Option 281 Opening the Sort Collection Chamber Door A Cell sorters that use droplet generation methods like the BD FACSAria II 5 M Tip can produce aerosols around the sample stream When acquiring biohazardous samples follow universal precautions at all times Keep the sort block door and the sort collection chamber door closed during sorting Follow these steps to stop sample flow and evacuate potential aerosols before opening the sort collection chamber door Use one of the following options to stop sample flow into the sort collection chamber e Click the Move Drawer button to close the aspirator drawer and sort sample into waste e Click the Acquire button to stop sample acquisition without unloading the sample tube
7. 204 BD FACSAria II User s Guide Changing the Integrated Nozzle Three sizes of nozzles are provided with your cytometer 70 85 and 100 um A 130 um nozzle can be ordered as an option The size is marked on the nozzle The closed loop nozzle used for cleaning and shutdown procedures is also changed with this procedure A A Any cytometer surface that comes in contact with biological specimens can 4 transmit potentially fatal disease Use universal precautions when handling sorting hardware Wear suitable protective clothing and gloves Turn off the stream and open the flow cell access door Turn the nozzle locking lever counter clockwise to the 6 00 position and pull the nozzle out of the cuvette flow cell Figure 6 20 Caution Do not open the sort block door while the nozzle locking lever is in the 6 00 position The flow cell can be damaged by the top of the door during opening Always turn the lever clockwise back to the 12 00 position before opening the sort block door Figure 6 20 Nozzle locking lever Nozzle locking lever in 12 00 position Insert the new nozzle into the flow cell with top side facing up and push it gently all the way forward until it stops Turn the nozzle locking lever clockwise to the 12 00 position Chapter 6 Shutdown and Maintenance 205 5 Turn on the stream and make sure it flows smoothly from the
8. Detector Array LP Mirror Replacement Laser PMI BP Filter Part No Intended Dye Trigon A 502 343797 405 nm violet laser 530 30 343798 Alexa Fluor 430 B 450 40 343801 Cascade Blue Pacific Blue Hoechst DAPI Alexa Fluor 405 The FSC photodiode is provided with a 1 0 ND filter Other filters are available as options Use the following part numbers to replace the filter Detector ND Filter Replacement Part No FSC photodiode 0 5 33710407 1 0 33710807 1 5 338651 2 0 338652 To order a filter holder use the following part numbers Holder Part No Mirror holder 336103 Mirror retainer 345662 Mirror backup spring 344979 Filter holder 336102 Filter retainer 333830 Appendix A Supplies and Consumables 257 Accessory Kit The cytometer 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 5 mL Eppendorf collection tube holder 644349 Four way 12 x 75 mm collection tube holder 641454 Two way 12 x 75 mm collection tube holder 641613 Two way 15 mL collection tube holder 641612 Universal top section for collection tube holders 644353 O ring for collection tube holder 3 337897 Integrated nozzles e 70 micr
9. cece cece eee e eens 310 Adjusting Area Scaling and Laser Delay 0 0 00 cece eens 310 Reusing the QC Experiment 0 0 c ce cee cece eee ence eens 322 Tracking OC Resultss sis 5 Sart 4 oot ee bats Das at Ba aS R 324 Index 325 xii BD FACSAria II User s Guide About This Guide This user s guide contains the instructions necessary to operate and maintain your BD FACSAria II flow cytometer Because many instrument functions are controlled by BD FACSDiva software this guide also contains basic software information needed for instrument setup To familiarize yourself with the software do the tutorials in the Getting Started with BD FACSDiva Software guide For detailed information on software features see the BD FACSDiva Software Reference Manual The BD FACSAria II User s Guide assumes you have a working knowledge of basic Microsoft Windows operation If you are not familiar with the Windows operating system see the documentation provided with your computer New users of the BD FACSAria II flow cytometer should read e Chapter 1 to become familiar with 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 opera
10. 100 micron 600 to 1 500 130 micron 400 to 1 200 Mi Tip 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 window Chapter 5 Sorting 159 5 Click Cancel in the Confirm dialog There is no need to collect the beads When the drawer is closed the beads are sorted to waste 6 Adjust the micrometer dial see Figure 1 21 on page 23 to obtain the brightest bead spot on the center stream 7 Click the Auto Delay button in the Side Stream window A dialog similar to Figure 5 5 on page 161 opens The graph in the left pane of the dialog represents brightness of the stream Y axis versus drop delay X axis Typically the graph should have a flat portion with several small peaks in either direction up or down or one prominent peak NOTE Ifthe original drop delay value was close to the appropriate value prior to starting auto delay then you may not get a graph displayed because few data points were required to determine the optimal drop delay 8 Select Start Run in the Auto Drop Delay dialog 9 Monitor the Auto Drop Delay dialog for progress A message appears when the process is completed NOTE If the sort is stopped during algorithm execution either by user action or because the system detected a failure the run will be stopped with an appropriate message Start the sort again verify that the s
11. The BD FACSAria II cytometer constantly applies drop drive energy 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 46 Patented Sweet Spot technology analyzes the drop breakoff image and provides feedback to the appropriate cytometer controls Chapter 2 Theory of Operation 45 46 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 Drop 1 and Gap FA 70 micron Stream a Sweet Spot x 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 Gap o gej 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 gr
12. beads 120 menu selections 68 overview 116 window 117 D daily shutdown procedure 176 Dashboard Acquisition 81 data analyzing 138 143 gating 137 143 recording 138 142 recording QC 319 data collection overview 131 default setup detector arrays 252 deflection drop 53 plates 15 152 153 removing 219 troubleshooting 234 deflection plate removal tool 219 delay drop 50 51 154 See also Accudrop laser 43 317 deleting custom devices 174 sort populations 91 sort precision modes 59 detectors 22 34 40 devices sorting 87 172 174 diode laser 23 51 discriminating filters 37 doors sort collection chamber 17 doublets discrimination experiment 97 discrimination gating 132 eliminating 142 drain plug 115 drawer See aspirator drawer drip tray 203 drop auto delay 159 breakoff 45 charging 52 conflicts 53 correction factors 51 154 deflection 53 delay 50 51 154 156 See also Accudrop drive frequency 47 formation 45 satellites 106 Drop 1 about 46 48 adjusting 48 106 E editing cytometer configurations 77 sort layouts 91 statistics view 144 electronics aborts 242 about 24 signal processing 41 troubleshooting 245 emergency stop button 25 emission optics 252 spectra 33 emptying waste 114 error messages cleaning mode failure 244 cytometer disconnected 245 cytometer not responding 245 Master DAQ overflow 245 ethanol shutdown filter changing 193 tank capacity 108 refilli
13. troubleshooting 235 connecting external air 4 fluidics cart 3 power cords 3 24 containers about 2 autoclaving 187 emptying waste 114 refilling 112 containment device sheath probe 186 controls See also buttons ACDU stage 92 acquisition 81 aspirator drawer 50 92 attenuation 50 compensation 134 cytometer software 63 flow rate 82 fluidics 63 optical filter 50 51 sorting 83 92 stream 47 Sweet Spot 47 test sort 50 tube loading 82 voltage 50 waste drawer 50 conventions user s guide xiv cooling samples 66 100 cord fluidics cart 3 5 counters sorting 93 creating analysis objects 143 application settings 123 compensation controls 134 custom devices 84 172 328 BD FACSAria II User s Guide experiments 311 folders 132 311 sort layouts 89 162 sort precision modes 59 statistics view 144 CS amp T See Cytometer Setup and Tracking custom devices 84 172 custom optical filters 222 customer support xv cuvette flow cell about 11 cleaning 176 cytometer about 6 adding configurations 74 cleaning 181 183 configuration 40 68 69 118 controls software 63 disconnect error 245 doors 7 electronics 24 fluidics 7 28 not responding 245 optics 18 performance 116 249 power 24 100 QC particles 261 quality control 308 shutting down 176 178 specifications 248 254 starting 100 status report 72 supplies 256 workstation 26 Cytometer Setup and Tracking CS amp T
14. 2nd Drop 17 a 3rd Drop 8 a 4th Drop 0 Phase 5 000 3 12 Click the Optical Filter button to move the emission filter away from the camera 13 Reset the window extension to its original setting typically 2 14 Turn off the deflection plates 158 BD FACSAria II User s Guide Determining the Drop Delay Automatic Method The auto drop delay feature automates setting the drop delay to get optimized results from sorting You should use this method after you have a good understanding of how drop delay works in general See Using Manual Drop Delay on page 156 for more information Overview of Auto Drop Delay Auto drop delay works best when the sort system is stable and assumes that BD Accudrop beads are used When the process is started the auto drop algorithm uses several passes to find best drop delay possible under current conditions of the system Coarse passes are used to find initial drop delay within 2 drops of the ideal The coarse passes are faster than fine tune passes Fine tune passes are used to locate the ideal drop delay value considering current conditions Using Auto Drop Delay 1 Set up an experiment for drop delay as described in Setting Up the Experiment on page 155 2 Loada tube filled with a suspension of BD Accudrop beads approximately 2 drops of beads in 0 5 mL PBS 3 Adjust the flow rate to achieve these values of events per second 70 micron 1 000 to 3 000 85 micron 800 to 2 000
15. E bama Na Fluorescence objective lens Nozzle 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 longpass 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 cytometer See Figure 1 19 on page 21 Figure 1 19 Transmission pathways in an octagon Chapter 1 Cytometer Components 21 22 Detectors A standard system is equipped with an octagon containing six PMTs that detect light from the 488 nm blue laser and a trigon containing two PMTs that detect light from the 633 nm red laser The third laser option adds an additional trigon containing two PMTs to detect light from the 405 nm violet laser If the optional 375 nm near UV laser is installed it uses the same PMTs as the violet 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 markers and two scatter parameters at a time Figure 1 20 Fully loaded detector arrays Trigons Blue laser signal
16. FSC A 1 000 Tube Name High Sort Setup Tube Name High Sort Setup Record Date Mar 28 2006 12 53 47 PM Record Date Mar 28 2006 12 51 14 PM SOP Administrator SOP Administrator FSC A FSC H FSC A FSC H Population Mean Mean Population Mean Mean ri 87 958 107 608 107 810 107 882 Appendix E QC Using BD FACSDiva Software 315 8 Adjust the FITC voltage to place the FITC H signal at approximately 100 x 10 a Click the Parameters tab in the Cytometer window b Select the FITC voltage field and adjust the voltage 9 Adjust the blue laser area scaling until the FITC A signal matches the FITC H signal if needed The blue laser area scaling can be adjusted using any parameter excited by the blue laser a Click the Laser tab in the Cytometer window b Adjust the area scaling factor for the blue laser Cytometer FACSAria 1 Cytometer Connected e If the FITC A signal is lower than FITC H increase area scaling e If the FITC A signal is higher than FITC H decrease area scaling 316 BD FACSAria II User s Guide Figure E 5 Blue laser area scaling before adjustment left and after right Rainbow Beads High Sort Setup Rainbow Beads High Sort Setup E as 3 a ad 2 zH 57 5 S34 Sg a a z 1 SAREE ASABE ARRAS RRS RE TTT PTT ery 100 150 200 280 50 100 150 200 25 FITC H amp 1 000 FITC H 1 000 2 pm Beads High Sort
17. Figure 2 11 Example BD FACSAria II cytometer configuration 2 laser 7 color 5 2 BD default Laser Blue 488nm FSC Laser Red 633nm APC CYT PE Texas Red 40 BD FACSAria II 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 Figure 2 12 Anatomy of a pulse O w signal intensity time signal intensity time O my signal intensity time A pulse begins when a particle enters the laser beam At this point both the beam intensity and signal intensity are low 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 As the particle leaves the beam the pulse trails off Chapter 2 Theory of Operation 41 42 Pulse Parameters A parameter is a pulse property that is generated by a single PMT or photodiode measuring fluorescent or scattered light You can measure three characteristics of a pulse area height and width
18. Open the sort block door Place 1 2 drops of DI water or ethanol on a cotton swab Click the Breakoff window and select Raw Image Visible smudges are more apparent in the raw image view While viewing the image on the Breakoff window insert the swab just below the bottom of the flow cell The strobe lens and upper camera windows are located behind two circular openings on either side of the top of the sort chamber You will see the end of the swab in the Breakoff window when you intercept either opening 218 BD FACSAria II User s Guide Strobe lens window Upper camera window 7 Gently wipe the upper camera window and then the strobe lens opposite the window to remove any saline 8 Repeat with isopropyl alcohol or 70 ethanol until clean Removing the Deflection Plates You can remove the deflection plates for cleaning by pulling the plates out using the deflection plate removal tool supplied in the accessory kit See Figure 6 32 Figure 6 32 Deflection plate removal tool Chapter 6 Shutdown and Maintenance 219 1 Make sure the deflection plates are turned off 2 Open the sort block door 3 Slide the deflection plate removal tool behind one of the plates and pull straight out from the sort block Hold your thumb on the plate or use your other hand as you pull it out so it does not fall as you remove it Figure 6 33 Using the deflection plate removal tool Lubricating the Sample Injection Chamber
19. Sample injection chamber does not close and causes a BISH or BISO error message O ring at bottom of sample injection chamber is dry and causing chamber to stick Lubricate the O ring and tube holder See Lubricating the Sample Injection Chamber O Ring on page 220 244 BD FACSAria II User s Guide Electronics Troubleshooting Observation Cytometer Disconnected in Cytometer window Possible Causes Cytometer power is off Recommended Solutions Turn on the cytometer main power Communication failure between workstation and cytometer e Exit the software and then restart it e If restarting does not work reset the cytometer 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 cytometer Ethernet cable between workstation and cytometer is disconnected 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 in Cytometer window Event rate is too high Decrease the event rate or verify the threshold Dirty flow cell Clean the flow cell See Cleaning the Flow Cell on page 176 Cytometer not Unknown Perform the suggestions for a responding in Status tab communication failure above Chapter 7 Troubleshootin
20. 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 BD 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 create a custom device See Creating a Custom Device on page 172 Use the following procedure to verify the home location and adjust it 1 Optimize the drop delay See Determining the Drop Delay 170 BD FACSAria II User s Guide Manual Method on page 154 Turn on the deflection plates Click the Voltage button in the Side Stream window The voltage warning light illuminates indicating that the plates are charged NOTE Make sure the center stream image does not move after the plates are turned on Major movement of the center stream could indicate that the plates or area around the plates needs cleaning See External Cleaning on page 181 Click the Test Sort button and optimize the far left side stream then click the Test Sort button again to turn it off 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 If you cannot see a stream image or the image is dim adjust the micrometer dia
21. 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 Verify that the sort precision mode is set to Initial See Sort Precision Modes on page 57 for more information 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 Chapter 5 Sorting 157 Figure 5 3 Sorting Accudrop beads in Initial mode FA 70 micron 42 J f f 91 Voltage Center 2nd Drop 20 3 3rd Drop 10 2 4th Drop o Phase 10 Inthe Sort Layout window change the precision mode to Fine Tune 11 Optimize the drop delay Adjust the drop delay value in 0 03 drop increments click arrow control until the left side stream intensity is greater than or equal to 90 Wait a few seconds after each click for a complete response to the delay change Figure 5 4 Sorting Accudrop beads in Fine Tune mode FA 70 micron Voltage Test Sort Optical Filter J Attenuation fq Waste Drawer Drop Delay 40 73 2 Auto Delay an l 21 g 100 voltage Center Plate voltage
22. e Click Unload to stop acquisition and unload the sample tube Press the up arrow button repeatedly to increase the suction to 100 Wait at least 30 seconds before opening the sort chamber doors to allow potentially hazardous aerosols to evacuate Place the system in standby by pressing the power button on the membrane panel of the evacuator Open the sort collection chamber door Remember to decrease the suction control rate to 20 when you return to sorting samples 282 BD FACSAria II User s Guide Turning Off the Evacuator Turn off the evacuator after you have finished running biohazardous samples 1 Place the system in standby by pressing the power button on the membrane panel of the evacuator 2 Switch off the main power on the back of the evacuator Figure C 4 on page 280 Maintenance Use the following guidelines to ensure optimal performance of the BD Aerosol Management Option Change the ULPA filter and attached tubing when the flow gauge indicator is gt 2 4 at a 20 flow setting or when the red filter life indicator LED is blinking See Replacing the ULPA Filter on page 284 Two spare filters and replacement tubing are included with the AMO To order additional replacement kits contact your local BD Biosciences representative A All biological specimens and materials coming into contact with them can transmit potentially fatal disease Handle the ULPA filter and attached tubing as if capable of transmit
23. s Guide e Sort Report Displays a report showing the sort settings acquisition counters and sort layout information from the current sort See Sort Report on page 94 Sort Setup Sort setup values for four different nozzle sizes can be downloaded using the Sort gt Sort Setup command Default settings are provided for each sorting option along with a custom setting for each option As a general rule for optimal results when sorting large or fragile cells use a larger nozzle size and lower pressure To increase throughput and yield when sorting smaller or less fragile cells use a smaller nozzle size and higher pressure Sort Setup Nozzle Default name microns pressure psi 70 micron 70 70 85 micron 85 45 100 micron 100 20 130 micron 130 10 If you make changes to any of the settings the changes are automatically saved when you exit 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 Default settings are shown in Table 3 2 on page 86 Chapter 3 Using BD FACSDiva Software 85 In addition the Sort Setup menu has following menu items 70 Custom 85 Custom 100 Custom and 130 Custom Initially all of these new setups are exact copies of the corresponding original setups with the same defaults This allows you to create two sets of sort values pressure for example for the same nozzle size A Do not use a nozzle size that is
24. snae Cell E 1 2 4 5 Sort location field for individual well View Counters BD FACSAria II User s Guide Figure 3 10 Sort layout for a frosted slide Tube_001 Sort Layout_001 Device Precision Target Events Save Sort Reports Save Conflicts CAGE v Single Cell w Ask User v 3 z Sort location B field for a spot on a slide altimiola Es Sort Rate Confl Cnt Confl Rate Efficiency View Counters Setting Up a Sort Layout Sort layouts can be added to tubes or global worksheets e Create tube specific layouts if you are sorting different populations or using different sort devices for each tube in the experiment e Create global worksheet specific layouts if you are sorting the same populations into the same sort device for all tubes in the experiment NOTE To create a tube specific sort layout make sure a normal worksheet white tab is shown in the Worksheet window before you create the layout 1 Select the icon for a tube or global worksheet in an open experiment and click the New Sort Layout button on the Browser toolbar 2 Inthe Sort Layout window select 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 t
25. to the recommended maintenance schedule Table 6 1 Scheduled Maintenance Procedure Recommended Frequency Internal Cleaning on this page See Table 6 2 on page 183 Purging the Fluid Filters on page 190 Weekly Purging the Sheath Filter on page 191 Weekly Changing the disposable waste cap see Monthly Emptying the Waste Container on page 114 Changing the Fluid Filters on page 191 Every 6 months Changing the Sheath Filter on page 192 Every 3 months Changing the Ethanol Shutdown Filter on page 193 Every 6 months Changing the Sample Lines on page 194 Every 4 6 months or as needed Changing the Air Filters on page 202 Every 6 12 months depending on cytometer use and the quality of the air Changing the Sheath Tank Air Filter on page 203 Every 6 months Checking the Fluidics Cart Drip Tray on page 203 Weekly 182 BD FACSAria II User s Guide Internal Cleaning BD FACSDiva software includes four pre programmed cleaning modes that can be used alone or in combination to provide the required level of cleaning The following sections describe the different cleaning modes See Table 6 2 on page 183 for an overview of each mode fava Sort Help Fluidics Startup Fluidics Shutdown Change Sample Filter Sheath Pressure Sample Agitation gt Sample Temperature gt Sample Line Backflush Clean Flow Cell Prime after Tank Refill Prepare
26. 29 2007 12 49 PM Cytometer Performance March 14 2007 03 34 PM Cytometer Performance Results Passed 4 Select File gt Exit to close the Cytometer Setup and Tracking window and connect back to the BD FACSDiva interface Chapter 4 Running Samples 121 5 Click Use CST Settings in the dialog that appears CST Mismatch The settings from CST are different from those on the cytometer A Do you want to use the CST values o Dont show this message again for current login sessiot Remember my dedsion mmm Details gt gt Use CST Settings Keep Current Settings By selecting Use CST Settings the laser delay area scaling and other cytometer settings will be updated to the latest optimized settings from the performance check Click the Details button to verify which cytometer settings will be updated At this point you can do one of the following e Continue to the next section to create application settings for an experiment you will use frequently e Collect data using existing application settings See Data Collection on page 131 122 BD FACSAria II User s Guide Application Settings Application settings are associated with a cytometer configuration and include the parameters needed for the application area scaling values PMT voltages and threshold values but not compensation Each time a performance check is run for a configuration the application settings associated with that configuration are updated
27. 3 Using BD FACSDiva Software 97 THIS PAGE INTENTIONALLY LEFT BLANK 4 Running Samples The following topics are covered in this chapter e Cytometer Startup on page 100 e Checking Cytometer Performance on page 116 e Application Settings on page 123 e Data Collection on page 131 e Data Recording and Analysis on page 138 99 Cytometer Startup Follow these steps to start up your BD FACSAria II cytometer 1 Start up the workstation 2 Turn on the cytometer main power Figure 4 1 Power panel H m Main power Main Power Blue Red Violet DOU If the cytometer was just shut down wait until the system is fully depressurized stops hissing before you turn on the main power 3 Turn on the lasers Wait 30 minutes for them to warm up To turn on a laser press the corresponding laser power button on the power panel Figure 4 1 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 66 100 BD FACSAria II User s Guide 4 Start BD FACSDiva software and log in with your user name and password To start the software double click the application shortcut on the desktop Le BD FACSDiva Software Check the fluidics levels in the Cytometer window Replenish fluids or empty the waste if needed To display the Cytometer window click the Cytomet
28. 4 Add the current month and year to the experiment name rename the specimen with the type of bead you are running such as Rainbow beads and rename the tube with today s date Adjusting Area Scaling for the Blue Laser 1 Open the QC experiment by double clicking the experiment icon Verify that the Area Scaling worksheet is displayed in the Worksheet window 2 Click to set the current tube pointer next to the tube with today s date in the Browser 8 44 Rainbow beads Current tube 6 022007 pointer 8S Cytometer Settings k Analysis 3 Install the tube of beads onto the loading port 4 Click Load in the Acquisition Dashboard 312 BD FACSAria II User s Guide 5 6 The loading port rises to enclose the tube within the chamber Once the tube is loaded acquisition starts automatically Set the Events to Display to 500 events EH Acquisition Dashboard Current Activity Active TubeYell Threshold Rate Stopping Gate Events Elapsed Time Blue 0 evtis 0 evt 00 00 00 Basic Controls j i BB Acquire Data i 8 Record Data estart Acquisition Setup Stopping Gate E AllEvents v Events To Record 10000evt v Stopping Time sec o iit Storage Gate LE Events 4 v Events To Display Csmet Dx Flow Rate 1 0 Sift Adjust the FSC and SSC voltages to place the particles within the P1 gate at approximately 100 x 10 on scale in the FSC H vs SSC H dot plot a Click the Parameters tab in the Cytomet
29. 9 336 100 0 7 Click Record in the Acquisition Dashboard to record data 8 When all events have been recorded click Unload and remove the tube from the cytometer 9 Install the next tube then click Next Tube in the Acquisition Dashboard to move the current tube pointer to the next tube 142 BD FACSAria II User s Guide 10 Repeat steps 7 through 9 for the remaining tubes 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 27 on page 145 1 Select Edit gt User Preferences 2 Inthe Gates tab set the preferences as follows User Preferences General Gates Worksheet Plot FCS Templates Statistics Biexponential Interval Gate Default Color O No color Has color Quadrant Gate Default Color O No color Same color for each quadrant 3 Create the following gates e Quadrant gate on the FITC vs PE plot e Interval gate on the APC vs PerCP Cy5 5 plot to capture the APC beads e Rectangle gate on the APC vs PerCP Cy5 5 plot to capture the PerCP Cy5 5 beads 4 Rename each population in the population hierarchy Mi Tip Press the Enter key twice to move to the next population without using the mouse Chapter 4 Running Samples 143 5 Right click either fluorescence plot and select Create Statistics View A statistics view is added t
30. Appendix E OC Using BD FACSDiva Software 309 Figure E 2 Error message showing details E CST Mismatch The settings from CST are different from those on the cytometer A Do you want to use the CST values o Don t show this message again for current login session Remember my decision No Detail Use CST Settings Keep Current Settings Details FSC Area Scaling CST 1 12 Current 0 99 Laser Area Scaling Red CST 1 37 Current 1 09 piue CST 47 CUECEnt 1 10 Violet CST 1 36 Current 1 05 Preparing QC Particles Prepare QC particles also known as beads in a 12 x 75 mm tube according to the manufacturer s instructions See Table E 1 for one brand of beads that can be ordered from BD Table E 1 Bead information BD FACSFlow SPHERO Rainbow Particles Ordering Info Solution 3 0 3 4 microns g 1 mL 2 to 3 drops BD Biosciences Cat No 556291 Adjusting Area Scaling and Laser Delay The required area scaling factor changes based on sheath pressure and particle size The area scaling factors should be verified for each experiment performed on the cytometer 310 BD FACSAria II User s Guide This section describes how to optimize area scaling and laser delay using the QC template that is included with BD FACSDiva software Setting Up the QC Experiment Follow the steps in this section to set up an experiment for instrument QC 1 Click the corresponding buttons on the Workspace toolbar to display the Br
31. Delay This section describes the manual method of optimizing the drop delay 1 3 4 Load a tube filled with a suspension of BD Accudrop beads approximately 1 2 drops of beads in 0 5 mL PBS In the Laser tab of the Cytometer window set the window extension to zero Cytometer FACSAria 1 BD Defaults ba Cytometer Connected Adjust the flow rate to achieve an event rate of 1 000 3 000 events per second Mi Tip If this cannot be achieved using a flow rate setting between 1 and 5 adjust the bead concentration Turn on the voltage in the Side Stream window Click Sort in the Sort Layout window 156 BD FACSAria II User s Guide 5 Click Cancel at the Confirm dialog P Confirm 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 and turn plates ON Click Cancel to start sort in current state There is no need to collect the beads When the drawer is closed the beads are sorted to waste Adjust the micrometer dial see Figure 1 21 on page 23 to obtain the brightest bead spot on the center stream Click the Optical Filter button 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
32. FACSAria II User s Guide Mi Tip Click Record Data to save data for the tube Acquisition and sorting continue after the required number of events has been recorded 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 Stop Acquiring button in the Dashboard or the Sort button in the Sort Layout window Monitor the sort progress from the Sort Layout window 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 Global Sheet1 Sort Layout _001 Device Precision Target Events Save Sort Reports Save Conflicts 4 way Purity v Ask User v Far Left Left Right Far Right B Fic 17752 J orc 20910 I Fercr cys 18389 Sort Rate Confl Cnt Confl Rate Efficiency ma View Counters NOTE When the Sweet Spot is on sorting pauses automatically if the Drop 1 or Gap values 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 n
33. If the Sweet Spot is on Drop 1 and target Gap values are shown BD FACSAria II User s Guide Figure 3 6 Cytometer Status Report ytometer Name FACSAriall Cytometer Status Report Date 2007 10 12 at 07 48 21 5 Number P94400001 tog Off Off On on on On On On On On on Spectral Overlap Channels f Colors Fire PE PE Texas Red PerCP Cy5 5 PE Cy7 APC APC H7 Pacific Blue AmCyan Sort Settings Sort Setup Frequency Amplitude Phase Drop Delay Attenuation Sweet Spot First Drop Target Gap Plates Voltage Voltage Centering Side Stream Voltage Far Left 80 00 Neighboring Drop Charge 2nd Chapter 3 Using BD FACSDiva Software 73 74 Custom Configurations Before you can record data you must first ensure that the cytometer configuration is appropriate for the experiment If needed you can create custom configurations to add parameters or filters and mirrors that are not listed in the base configuration You will also need to create custom configurations for each sheath pressure you would like to run When creating custom configurations use descriptive names that make it easy to identify the configuration See the BD FACSDiva Software Reference Manual and the BD Cytometer Setup and Tracking Application Guide for more information Preparing for Custom Configurations Custom configurations can only be created by users with administrator access The easiest way to cr
34. Mix the beads by gently inverting the vial 2 In a 12 x 75 mm tube add 0 35 mL of sheath fluid and 1 drop of beads NOTE Beads are stable at 2 C to 25 C for no more than 20 minutes if stored in direct light and up to 8 hours if protected from light Running a Performance Check The performance check feature of Cytometer Setup and Tracking will check the cytometer s daily performance 1 Install the bead tube onto the cytometer loading port 2 Inthe Setup Control window select Check Performance from the Characterize menu 3 Click Run Setup Control RUO Load a tube with beads and click Run button to start setup Characterize Check Performance v Y Plots appear under the Setup tab and the performance check is run The performance check takes approximately 5 minutes to complete 120 BD FACSAria II User s Guide Reviewing the Results 1 Once the performance check is complete click View Report 2 Print the report 3 Verify that the performance check passed In the Setup tab the Cytometer Performance Results should have a green checkmark and the word Passed next to it If any parameters did not pass see the BD Cytometer Setup and Tracking Application Guide for troubleshooting information File Cytometer Tools Setup Reports Performance Tracking System Summary OK Cytometer Configuration 3 laser 9 color 5 2 2 Configuration 5 BD default Lot ID 68341 Cytometer Baseline January
35. 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 area 64000 intensity gt signal pulse height height You can select which pulse characteristics you lt gt time gt l want to measure for each parameter using the width Parameters tab 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 the 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 Cytometer FACSAria 1 Status Parameters Threshold Laser Cc eae ao Parameter Voltage Cytometer Connected Digital data is displayed on an 18 bit linear scale from 2 6 262 144 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 See the BD FACSDiva Software Reference Manual for more information BD FACSAria II User s Guide Laser Delay Sample interrogation takes place within the cuvette flow cell Fiber optic cables are used to direct laser light through a series of prisms tha
36. Troubleshooting the Stream continued Observation Leaking or spraying around nozzle Possible Causes Defective or damaged integrated nozzle seal Recommended Solutions Replace the defective seal with a standard O ring Temporary Replacement of a Seal on page 208 Defective or damaged O ring in standard nozzle Replace the O ring See Installing an O ring in a Standard Nozzle on page 209 Nozzle inserted improperly Turn off the stream Remove the nozzle See Changing the Integrated Nozzle on page 205 for instructions Extra O ring is blocking the nozzle Remove the nozzle and use a cotton swab to clear out the cuvette Drop breakoff is 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 is on Turn off attenuation Amplitude is 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 seal or O ring is in place Re insert the nozzle and slide the nozzle in until it stops then close the locking lever Chapter 7 Troubleshooting 229 Troubleshooting the Stream continued Observation Possible Causes Recommended
37. Tubing loose or not connected Ensure that the tubing is securely connected below the sort chamber and to the filter module Tubing kinked or damaged Inspect the tubing for kinks or punctures Replace the tubing if needed Wrong tubing type or part Ensure that the correct type of tubing is in use Erratic reading on filter flow gauge Defective filter Replace the filter Filter improperly seated in evacuator Reseat the filter with the evacuator power off Off scale reading on filter flow gauge Tubing or sort collection chamber obstructed e Inspect the tubing for kinks or punctures Replace the tubing if needed e Check for obstructions in the sort collection chamber Remove any obstruction Filter clogged or saturated Replace the filter as described on page 284 292 BD FACSAria II User s Guide Specifications Specifications for the BD Aerosol Management Option are as follows Evacuator e Greater than or equal to 7 CFM ft3 min normal operation rate 20 suction control e Greater than or equal to 30 CFM boost evacuation 100 suction control e lt 35 lb unpacked weight ULPA Filter Module e VLSI grade e Traps particles greater than or equal to 0 12 pm e Three stage filtration pre filter ULPA post filter e Particulate removal efficiency gt 99 9995 Air Filter e Filter media is an open cell polyurethane foam e Media specially coated for improved fire reta
38. about this feature see the BD FACSDiva Software Reference Manual If you are performing compensation manually not all steps will apply Setting Up the Workspace Before you begin optimizing settings it is important to verify the cytometer configuration and create an experiment containing appropriate parameters for the assay 1 Select Cytometer gt View Configurations and verify that the current configuration is appropriate for the type of sample you are running 2 Verify that there is a valid performance check for the configuration Chapter 4 Running Samples 131 3 Make sure the octagon and trigon s contain appropriate filters For assistance see Cytometer Configuration on page 68 A For accurate data results the cytometer optics must match the current cytometer configuration 4 Click the corresponding buttons on the Workspace toolbar to display the Browser i Cytometer Inspector P1 Worksheet E and Acquisition Dashboard windows as needed 5 Optional Create a folder for your experiment Select the icon for your database and press Ctrl N Rename the folder appropriately See the BD FACSDiva Software Reference Manual for ideas on how to organize experiments Vv Tip To place an experiment inside a folder select the folder before you create the experiment 6 Create an experiment from the Doublet Discrimination Gating template For best results we recommend that you perform doublet discrimina
39. are determining the drop delay The button changes to red 7 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 stream and the fewest in the center stream Chapter 2 Theory of Operation 51 52 See Determining the Drop Delay Manual Method on page 154 for more information Auto Drop Delay The Auto Drop Delay feature enables the system to optimize the drop delay automatically See Determining the Drop Delay Automatic Method on page 159 Drop Charging Drops are charged when an event is detected and meets the defined sorting criteria as specified in the Sort Layout window The Sort Layout window contains all sorting instructions and controls Figure 2 16 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 87 for more information about the Sort Layout window Figure 2 16 Sort layout for a four way sort Global Sheet1 Sort Layout_001 Device 4 Way Purity v Precision Target Events Save Sort Reports Save Conflicts Ask User v Far Left Left
40. be defined by up to eight gates The following topics are covered in this chapter e Setting Up for Sorting on page 150 e Determining the Drop Delay Manual Method on page 154 e Determining the Drop Delay Automatic Method on page 159 e Sorting on page 161 e Setting Up for Sorting Into a Plate or Slide on page 168 Before you begin you should be familiar with BD FACSAria II operation using BD FACSDiva software Review chapters 2 and 3 if needed 149 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 Start up the cytometer and the computer See Cytometer Startup on page 100 Install the appropriate size nozzle Select a new sort setup mode if needed and select an appropriate cytometer configuration To change your current sort setup mode select a nozzle size from the Sort gt Sort Setup menu For more information see Sort Setup on page 85 Install a sample line filter if needed See Installing or Removing a Sample Line Filter on page 212 Check the laser delay for your sheath pressure and particle size e To set the laser delay automatically using the Cytometer Setup and Tracking module see Checking Cytometer Performance on page 116 e To set the laser delay manually see QC Using BD FACSDiva Software on page 307 Optimize cytometer settings for the sample to be sorted See Data Collection
41. bypass tubing install the filter and repeat the priming procedure one last time Communication failure between workstation and cytometer Exit the software and restart it Chapter 7 Troubleshooting 227 Troubleshooting the Stream continued Observation Possible Causes No stream when Stream Sheath tank low or empty control clicked Recommended Solutions Refill the sheath tank See Refilling the Sheath Tank on page 109 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 184 Air in sheath filter Purge the sheath filter See Purging the Sheath Filter on page 191 Dry filter Open the bleeder valve to purge the filter See Purging the Fluid Filters on page 190 Fanning around center Nozzle inserted improperly stream Re insert 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 10 seconds Click the Stream control on then off Sonicate the nozzle and re install It Fluid line connected to ethanol shutdown tank Move fluid and air lines to sheath tank then perform Fluidics Startup See Performing Fluidics Startup on page 101 228 BD FACSAria II User s Guide
42. channel is too low Adjust the threshold channel See Calculating Compensation on page 135 Sample is too concentrated Dilute the sample Flow rate is too high Decrease the flow rate in the Acquisition Dashboard Bubbles in flow cell Turn off the stream wait a few seconds and turn on the stream again Unexpectedly low event rate Sample not adequately mixed Increase the sample agitation rate See Sample Agitation on page 65 Threshold channel is too high Adjust the threshold channel See Calculating Compensation on page 135 Sample is too dilute Concentrate the sample Sample line is clogged or kinked Backflush the sample line See Sample Line Backflush on page 184 If necessary change the sample line Look for visible kinks in the line If kinks are found change the sample line See Changing the Sample Lines on page 194 240 BD FACSAria II User s Guide Acquisition Troubleshooting continued Observation Unexpectedly low event rate continued Possible Causes Sample line installed incorrectly Recommended Solutions Verify the sample line installation See Changing the Sample Lines on page 194 Sample aggregates Filter the sample Memory is full Compare the processed event rate in BD FACSDiva software with the threshold counter If the event rate is much lower exit and then restart the application Distorted populations
43. configuration from the list 118 BD FACSAria II User s Guide Configurations Parameters Filters and Mirrors amp Base Configurations Training class configurations 70 35 Medium 100 20 Low E 3 laser 9 color 5 2 2 Configuration 5 01 02 07 E 70 70 High 04 04 07 04 04 07 04 04 07 c Click Set Configuration Click OK Verify that the current configuration has a valid baseline defined If not see the BD Cytometer Setup and Tracking Application Guide for more information on running a baseline Cytometer Setup and Tracking Eile Cytometer Tools Setup Reports Performance Tracking Valid baseline Expired performance check System Summary Requires Attention Cytometer Configuration FACSAria 5 2 2 Violet High Sort Setup Lot ID 68341 Cytometer Baseline January 29 2007 01 33 PM a Cytometer Performance January 31 2007 01 50 PM Cytometer Performance is expired We recommend Cytometer Performance Results Passed Chapter 4 Running Samples 119 Preparing the CS amp T Beads The BD Cytometer Setup and Tracking beads consist of bright mid and dim beads dyed with a mixture of fluorochromes Use the beads to define a baseline and to check cytometer performance using the CS amp T application See the BD Cytometer Setup and Tracking beads package insert for more instructions about preparing the bead suspension 1
44. for Aseptic Sort Table 6 2 Cytometer cleaning modes Cleaning Mode Summary Frequency Sample Line Backflush on page 184 Flushes the sample line with sheath fluid After running samples with adherent cells or dye Cleaning the Flow Cell on page 176 Cleans the sample path and the flow cell with DI water When indicated by distorted scatter or high CVs or as a daily shutdown procedure Prime After Tank Refill on page 184 Primes the fluid lines for the designated fluid s When a fluidics line is unplugged to refill a tank Prepare for Aseptic Sort on page 187 Decontaminates the complete sheath path and sample path with bleach DI water and ethanol When needed before aseptic sorting Chapter 6 Shutdown and Maintenance 183 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 is unloaded Perform the sample line backflush when you observe sample carryover or after running samples with adherent cells or dye NOTE Keep the stream running while performing the backflush 1 Select Cytometer gt Cleaning Modes gt Sample Line Backflush 2 Click Start to start the backflush Sample L
45. gating strategy Sorting parent and child populations into two different tubes 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 236 BD FACSAria II User s Guide Acquisition Troubleshooting Observation No events in plots after clicking Load or Acquire Data Possible Causes Current tube pointer is not set to current tube Recommended Solutions Click to move the current tube pointer to the appropriate tube Laser shutter is engaged Make sure the flow cell access door is completely closed Laser power is off Turn on the laser power Laser delay set incorrectly Adjust the laser delay settings See Cytometer Quality Control Using BD FACSDiva Software on page 308 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 select Show Populations Verify that the appropriate populations are displayed Uncolored events in plot 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 cytometer configuration di
46. groove 1 Use the wooden end of a cotton swab or similar tool to install the O ring in the nozzle groove then allow the nozzle to air dry for a few minutes Do not wipe the nozzle with anything because it could leave fibers or other contamination or dislodge the O ring Use the magnifying glass in the accessory kit or a microscope to inspect the nozzle to verify the O ring is installed all the way into in the groove Chapter 6 Shutdown and Maintenance 209 Closed Loop Nozzle Maintenance The closed loop nozzle and related tubing should be cleaned if there are any indications of clogging or kinked tubing Cleaning the Integrated Closed Loop Nozzle 1 2 Turn off the stream and open the flow cell access door Remove the closed loop nozzle from the cuvette flow cell Turn the nozzle locking lever counter clockwise to the 6 00 position Remove the nozzle by pulling it straight out See Figure 6 22 Figure 6 22 Closed loop nozzle Unscrew the nut on the side of the nozzle to remove the tubing Make sure the ferrule stays on the tubing as you remove it Sonicate the nozzle for approximately 1 minute Sonicate the nozzle in a test tube containing DI water or a mild detergent Repeat the sonication as needed until the nozzle is clean Make sure the ferrule is on the tubing then screw the nut back into the hole in the side of the closed loop nozzle See Figure 6 23 210 BD FACSAria II User s Guide Replacing the
47. installed see the next section This procedure fills the flow cell with cleaning solution This is normally sufficient to keep the flow cell clean and operating properly If the system is used to process many different sample types or the system has problems with contamination you can perform a more extensive cleaning by running the Fluidics Shutdown command See Fluidics Shutdown on page 178 In addition to one of the choices listed above you should also perform an external cleaning See External Cleaning on page 181 Cleaning the Flow Cell Use the Clean Flow Cell command to run a tube of DI water through the sample line and flow cell This procedure can be used in two ways e As the daily shutdown procedure e Any time poor optical performance indicates that additional cleaning is needed NOTE After the procedure is complete the DI water remains in the flow cell until the stream is restarted To clean the flow cell 1 Turn off the stream 2 Remove the nozzle and install the integrated closed loop nozzle See Figure 6 1 on page 179 If you are using a standard closed loop nozzle verify that there is an O ring in the nozzle before installing it 3 Select Cytometer gt Cleaning Modes gt Clean Flow Cell 176 BD FACSAria II User s Guide When prompted install a tube containing approximately 3 mL of sterile filtered DI water then click OK Confirm Install a tube with cleaning solution on the loading po
48. le mat riel brouilleur du Canada History Revision Date Change Made 643245 12 07 Initial release 644832 3 09 Revised to include integrated nozzles plus additional changes Contents About This Guide xiii CONVENTOS ou fe Va ae BA ees A Os eee ty A eel SA ad Sse xiv Technical Assistance siame een oa hae a a SG E aae i aa XV Limitations se eea ne merean ae ne e e ea a a a a E a a bees xvi Chapter 1 Cytometer Components 1 Fluidics Cart erate ees bate SEAS ee bad ENO Ea a aE ieee Oe 2 Containers and Connectors 0 e cece cece ence n ene nees 2 Connecting to an External Air Supply 0 ce eee eee eee eee ee 4 Power and Operation ressora ir irita Poke eae La ee 5 Blow Gytomietert sors Scasdecack an see oe ed ee cae ade Ae ce eee ae Aa See a ad 6 Fluidics Components i vars beds Gadad Sees ieaa fied boda bbs Sak 7 Optics System ios os oe be eae ea See bl bee a Oeeie E Oe ee be a Ea 18 Cytometer Electronics 2 ccn cs acack dea baa ie de a dees ba ae 24 Emergency Stop Button rores eeraa radi Eea eee eee ee eee 25 Workstation seui st ici annette ee neha leu wa gdlnuls EEEREN ahe eked 26 Chapter 2 Theory of Operation 27 Fluid Movement a sda int ce eng eee E Pek awters Wikia Ate WONG Mie 28 Sheath Flow 4 0 0 3 Sta ea too sg Sake we aa ee OE ees eS ee es 29 Sample Flown aed ance o EE ahaa oak aes Moe ea 30 Signal Generation erisa pend a chad e repra te baba adian peur bbaih dancin 32 Light Sca
49. 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 8 7 on page 252 BD FACSAria II User s Guide Bandpass Filters 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 Figure 2 9 Light transmittance by bandpass filters 100 Bandpass 460 500 540 480 520 S 2 E 50 oO x 0 450 500 550 Wavelength nm 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
50. nozzle into the center of the waste aspirator If the stream is flowing but the breakoff is too long or the gap is unsteady this could indicate that there are bubbles in the flow cell If these conditions occur turn off the stream wait for 10 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 see Troubleshooting the Stream on page 226 NOTE After changing the nozzle you might need to adjust the angle of the sort block to re center the stream in the aspirator To do so loosen the adjustment screws on both sides of the deflection plates and rotate the sort block see Figure 1 12 on page 14 Tighten the screws when the stream is centered in the aspirator For further assistance see Troubleshooting the Stream on page 226 Cleaning the Integrated Nozzle Use the following procedure to clean the nozzle when the stream appears blocked or distorted To verify that the nozzle is clogged examine the opening at the center of the seal area under a microscope Figure 6 21 shows an example of an unclogged nozzle tip Figure 6 21 Magnified view of an integrated nozzle tip Seal in integrated nozzle A A All biological specimens and materials coming into contact with them can N transmit potentially fatal disease Handle nozzles as if capable of transmitting infection Wear suitable protective clothing and gloves 206 BD FACSAria II User s Guide To c
51. or filter not completely seated Make sure the appropriate filter is installed for each fluorochrome Make sure the filters are pushed all the way in Laser delay is set incorrectly Adjust the laser delay settings See Cytometer Quality Control Using BD FACSDiva Software on page 308 238 BD FACSAria II User s Guide Acquisition Troubleshooting continued Observation Possible Causes Recommended Solutions Low area signal Area scaling is too low Adjust area scaling for the corresponding laser See Cytometer Quality Control Using BD FACSDiva Software on page 308 Unexpected events in Incorrect logic in population Verify the gating strategy plot hierarchy Incorrect population s in Right click the plot and select plot Show Populations Verify that the appropriate populations are displayed Incorrect drawing order Verify that the required population is not hidden by another population Right click the plot and select Order Populations by Count Chapter 7 Troubleshooting 239 Acquisition Troubleshooting continued Observation Erratic event rate Possible Causes Sample aggregates Recommended Solutions Filter the sample Bulk injection O ring is worn Contact your BD Biosciences service engineer Sample is contaminated Re stain the sample making sure the tube is clean Sheath tank low Fill the sheath container Unexpectedly high event rate Threshold
52. outside of the purity mask so the interrogated drop would be sorted See Figure 2 21 Figure 2 21 Non target particle outside a purity mask of 8 Trailing drop Drop being interrogated Leading drop Naa Ly Purity mask Sorted Purity mask Non target particle With any purity mask greater than zero the drop being interrogated must be free of contaminating particles or the drop will not be sorted If the purity mask is set to zero a droplet containing the event of interest will be sorted regardless of contaminating particles Chapter 2 Theory of Operation S55 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 See Figure 2 22 Figure 2 22 Sorted and unsorted drop with phase mask of 16 Trailing drop Drop sorted Leading drop L Phase mask Trailing drop Drop not sorted Leading drop L LC Phase mask Decreasing the phase mask to 8 allows more dro
53. requires a commercial sublicense from Amersham Biosciences Corp 800 Centennial Avenue Piscataway NJ 08855 1327 USA Class I 1 Laser Product For Research Use Only Not for use in diagnostic or therapeutic procedures Patents APC Cy7 US 5 714 386 BD FACS Accudrop 6 372 506 Sweet Spot 5 700 692 FCC Information WARNING Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment NOTICE This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his or her own expense Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits This Class A digital apparatus meets all requirements of the Canadian Interference Causing Equipment Regulations Cet appareil num rique de la classe A respecte toutes les exigences du R glement sur
54. slot The sample line can drip if the pinch tubing is not all the way into the slot in the pinch valve 216 BD FACSAria II User s Guide Cleaning the Camera Windows Lower Camera Window 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 Figure 6 31 Lower camera and diode laser windows Diode laser window Lower camera window Upper Camera Window 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 Chapter 6 Shutdown and Maintenance 217 components after a clog or after sheath fluid has leaked or sprayed Follow the steps in this section to clean the strobe lens and upper camera window A amp Ava 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 open To clean the strobe lens and upper camera window 1 Ensure that the deflection plates are turned off warning light is not illuminated Turn off the stream
55. 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 global worksheets Sort layouts are available for up to nine default collection devices shown in the following figure Additional custom devices can be defined See Creating a Custom Device on page 172 6 Well Falcon 24 Well Falcon 48 Well Falcon 96 Well Falcon 384 Well Falcon Slide Frosted End Slide Standard Default collection devices Chapter 3 Using BD FACSDiva Software 87 88 Examples of sort layouts for different devices are shown in the following figures Instructions for setting up a sort layout can be found in Setting Up a Sort Layout on page 89 Figure 3 9 Sort layout for collection tubes top and for a 48 well plate bottom Tube_001 Sort Layout_001 Target Events Save Sort Reports Save Conflicts Collection 4 Tube it v Ask User x device Far Right Far Right Sort location Sort Sort Rate A field for counters far right tube Sorting Sol m Pa View Counters controls amp Tube_001 Sort Layout_001 Device Precision Target Events Save Sort Reports Save Conflicts E Well Falcon v
56. sort location is the well or spot on the lower right corner of the collection device 7 Click Apply then Close After you set the home and farthest locations custom devices are listed in the Device menu in the Sort Layout window NOTE Once custom devices are defined you cannot change the number of rows and columns 8 Click the Voltage button to turn off the deflection plates 9 Proceed with Determining the Drop Delay Manual Method on page 154 and Sorting on page 161 Deleting a Custom Device 1 Select Sort gt Custom Devices 2 Select the name of the custom device to be deleted in the Custom Devices dialog 3 Click Delete The device is deleted from the Custom Device list but is retained within any sort layouts where it was used 174 BD FACSAria II User s Guide 6 Shutdown and Maintenance The BD FACSAria II cytometer is designed to require minimum maintenance However to preserve the reliability of the cytometer you must regularly perform basic preventive maintenance procedures This chapter explains routine maintenance procedures you should follow to keep your cytometer in good condition Maintenance procedures are presented as follows e Daily Shutdown on page 176 e Scheduled Maintenance on page 182 e Unscheduled Maintenance on page 204 175 Daily Shutdown In the BD FACSAria II system the recommended daily shutdown procedure is to run the Clean Flow Cell command with the closed loop nozzle
57. 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 6 ACDU stage set up with plate left and slide right dn JAIS SIHL Well A1 Sorting starts on the front left corner of the device A1 location and proceeds from front to back and then from left to right 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 of the slide Make sure that you set up your sort layout accordingly See Figure 5 7 on page 170 Chapter 5 Sorting 169 Figure 5 7 Sort order on a slide OVOTO OO0O0O00 OJO JOOOO0O00 oqo OO0O000 Tube_001 Sort Layout_001 Device Precision Slide Frosted End v Single Cell v Target Events Save Sort Reports Save Conflicts w Ask User v 1 3 nloj afmjo G H I Sort Rate Confl Cnt Confl Rate Efficiency Setting Up the Stream View Counters 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 pre programmed to move a set distance between wells on a plate or spots on a slide
58. the loading port and place a tube in the holder Make sure to press the tube holder down firmly onto the metal rod in the loading port so the tube holder is seated correctly each time a tube is installed When the Load button is clicked in the software see Acquisition Controls on page 81 the loading port rises to enclose the tube within the chamber Figure 1 9 Tube holders eee 15 ml 12 X 75 mm oi e od Oo D A Tube holders 1 mL microtube After a tube is loaded the Load button changes to Unload Click 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 potential sample carryover A To prevent injury from moving parts keep your hands and clothing away from the loading port when a tube is loading or unloading Do not place objects under the loading port 10 BD FACSAria II User s Guide Cuvette Flow Cell The cuvette flow cell is the heart of the BD FACSAria II cytometer Figure 1 10 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 Figure 1 10 Cuvette flow cell Flow cell Laser beams Qe O f AAE ro ay Interrogation point M e heer o P
59. the plates needs cleaning 4 Click the Test Sort button and optimize the side streams See Figure 5 2 Adjust the voltage sliders to view the required number of streams Figure 5 2 Turning on Test Sort FA 70 micron Test Sort button J Voltage sliders voltage Center f Plate Voltage 5 000 ag 2nd Drop 20 4 3rd Drop 10 zZ 4th Drop o zZ Phase Chapter 5 Sorting 153 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 21 on page 23 5 Adjust the 2nd 3rd and 4th Drop settings to tighten the center 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 each collection tube a In the Side Stream window click the Waste Drawer button to open the drawer b Open the sort block door and aim each side stream into the tube as you adjust the corresponding slider in the Side Stream window c When you are satisfied with the side stream deflection close the sort block door 7 Click the Voltage button to turn off the deflection plates Determining the Drop Delay Manual Method BD FACS Accudrop technology is used to determine the optimal drop delay setting for your sorting application For more information see Drop Delay Overview on p
60. third 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 See Table 3 2 on page 86 for starting values e If the amplitude is lt 10 volts turn on attenuation in the Side Stream window See Table 2 2 on page 50 Chapter 4 Running Samples 105 2 Verify that the small satellite droplets are Good breakoff Poor breakoff merging with the large droplets FA 70 micron X J FA 70 micron x Stream Satellite merging is largely dependent on nozzle Sweet Spot position If the satellites are not merging you might need to remove and reinstall the nozzle or clean it by sonicating The satellite drops should merge into the drops in 6 satellites or fewer 3 Enter 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 need to reset it unless you change the nozzle or your sort setup option Vv Tip If the target value causes the amplitude to exceed 70 volts perform the recommendations under step 1 and re enter the Drop 1 target value 4 Turn on the Sweet Spot when the drop pattern is stable The Sweet Spot is designed to automatically adjust the drop drive a
61. to protect the sort collection tubes e A warning message appears on the screen Chapter 1 Cytometer Components 25 NOTE The emergency stop button does not turn off the lasers or shut down the cytometer main power Do not reset the button until the message appears To reset the button turn the button clockwise until the light turns off and the button returns to its original position Workstation Data acquisition and analysis as well as most BD FACSAria II cytometer functions are controlled by BD FACSDiva software on a third party PC 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 operating system e Microsoft Office application package e BD FACSDiva software version 6 1 or later Data acquisition and analysis Automatic fluidics startup shutdown and cleaning modes Sort setup stream monitoring clog detection and recovery CS amp T module for setup and tracking performance e Supporting documentation for the software For information about software features specific to the BD FACSAria II see Chapter 2 Theory of Operation and Chapter 3 Using BD FACSDiva Software For general software information See the BD FACSDiva Software Reference Manual Mi Tip For easy access to the online BD FACSDiva Software Reference Manual select Help gt Literature gt Reference Manual 26 BD FACSAria II User
62. to the latest run Using application settings provides an easy consistent and reproducible way to reuse cytometer settings for your commonly used applications See the BD Cytometer Setup and Tracking Application Guide for information on running a baseline and for other details on using the CS amp T application Before creating application settings you must e Perform the cytometer startup procedure described in Cytometer Startup on page 100 e Runa performance check for the cytometer configuration that will be used for the application Creating Application Settings This section describes how to create and save application settings for a multi color immunophenotyping sample Setting Up the Workspace 1 Create a new folder 2 Create a new experiment 3 Select the Cytometer Settings in the Browser 4 Inthe Inspector window click the Parameters tab and delete any unneeded parameters Chapter 4 Running Samples 123 Select the H checkbox to select height for each parameter See Figure 4 13 on page 124 Figure 4 13 Inspector window Parameters tab Inspector Cytometer Settings Cytometer Settings Parameters Threshold Ratio Compensation Parameter Voltage e FSC 402 ssc e FITC PIPE APC Delete Adjusting Area Scaling The required area scaling factor changes based on sheath pressure and particle size The ar
63. 0 Current 1 02 Laser Delay FSC Red CST 40 66 Current 41 37 Wolet 375 UY CST 34 63 Current 33 96 Laser Delay PMT Red CST 39 77 Current 40 59 Violet 375 UV CST 35 5 Current 34 63 Laser Area Scaling Red CST 0 99 Current 0 98 Blue CST 1 15 Current 1 14 Violet 375 UY CST 1 02 Current 0 93 Acquisition Controls Along with the controls described in the BD FACSDiva Software Reference Manual the following acquisition controls are available for the BD FACSAria II cytometer HE Acquisition Dashboard Current Activity Active Tube Vell Threshold Rate Stopping Gate Events Elapsed Time Tube oT Jo evtis 0 evt 00 00 00 Basic Controls Acquistion Setup Stopping Gate BB Ai Events v Events To Record 10000evt v Stopping Time sec o Sf Storage Gate MB At Events v Events To Display 1000 evt v Flow Rate 1 0 St Acquistion Status Processed Events Threshold Count Chapter 3 Using BD FACSDiva Software 81 82 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 NOTE The Load button is enabled only when the workstation is connected to the cytometer an experiment is open the stream is turned on and the current tube poin
64. 00 Window Extension us 2 00 Nozzle Size p 70 v Mi Tip The name of the configuration is displayed at the top of the BD FACSDiva workspace If you use a descriptive name for each configuration such as 100 15 Custom for nozzle size sheath pressure sort setup mode it will be easier to verify that the appropriate settings match without going back to view the configuration BD FACSDiva Software Administrator 70 70 PerCP conf 5 File Edit View Experiment Populations Worksheet Cytometer Sort Help Configuration Mismatch Dialog If there is a mismatch between the CS amp T settings and those in BD FACSDiva software a dialog opens Figure 3 7 on page 81 to indicate what the differences are and gives these options e Use CS amp T Settings Select this option if you have run a performance check or created a new configuration and want to use those values e Keep Current Settings Select this option if you were viewing a configuration in CS amp T and don t want to change the values For example settings for area scaling for the particle of interest 80 BD FACSAria II User s Guide Figure 3 7 CS amp T Mismatch Message The settings from CST are different from those on the cytometer Do you want to use the CST values Dorit show this message again for current login session Remember my decision Details Sheath Pressure CST 20 0 Current 70 0 Sort Mode CST 100 micron Currente70 micron FSC Area Scaling CST 1
65. 1 FITC A PE A PerCP APC A x PerCP Cy5 5 i b a Population Events Parent Mean Mean Mean Mean D Gre 228 2 4 246 6 787 28 32 Fon Bric 205 2 2 27 502 49 9 29 o7 Barc 240 2 6 173 41 22 14 781 2 WB Percp cy5 5 172 1 8 242 43 2 300 27 Chapter 4 Running Samples 145 Performing a Batch Analysis Batch analysis allows you to automatically advance through a selected set of tube data when using a global worksheet To perform a batch analysis 1 Verify that the global worksheet you will be using for analysis is displayed in the worksheet window 2 Right click the specimen to analyze in the Browser and select Batch Analysis The Batch Analysis dialog appears Only tubes under the selected specimen will be processed Tubes without data are skipped during a batch analysis 3 Do the following in the Batch Analysis dialog Batch Analysis v Output To Printer Statistics v View Time 5 Y Save as PDF V Freeze Biexponential Scales Manual Use Preferred Global Worksheet Export Filename tistics Batch_Analysis_02032007173318 csv Status 0 e Select Auto to analyze all the files without user intervention e Select 5 from the View Time menu to pause the analysis for 5 seconds after each tube is loaded e Select the Output to Printer checkbox to print a copy of the analysis after the data for each tube is loaded 146 BD FACSAria II Use
66. 1 Workspace Components When you start BD FACSDiva software the workspace appears See Figure 3 1 For a general overview of the workspace and to get started using the software see Getting Started with BD FACSDiva Software When running BD FACSDiva with the BD FACSAria II two additional windows can be displayed by clicking the Sorting button on the Workspace toolbar See Sorting Controls on page 83 for a description Figure 3 1 BD FACSDiva workspace BD FACSDiva Software Administrator Fie Tat Vew Experment Pupdatons Worksheet 4 aapne n CE Brinar Experiment _001 X O Cytoonetier FACSAria 1 x E Guhel Worksheet Ginkal Sheet Bot ree uUsArase g ge D E S Esta tenes Tube Latak aoa keywords Nare Tibe 00i Gobel Peet Tota of Everts Raard Date OF Ae quisitiun Dashtnard Current aemey Acw Tuevell Trresns Rate Stopping Gate Evonte Elapsed Tene Ue GoTo evtis Oevt 00 00 00 Bam Contra of met Tube Boxe Bi Record kasen stu Stopprg Gate AA rverts w Cvents FoRecod 10000evt w Stopping Tine op Storage Gate teres x terts tobair Soot w Fowrate 1 0 BI tegiren abe Dactrorie Abort Rates Bectronie Abort Count Concent O Picks atip dove 62 BD FACSAria II User s Guide Cytometer Controls Most BD FACSAria I specific cytometer controls are cytometer ae accessed through the Cytometer menu Controls on the Fluidics Startup Fluidics Shutdown Cytometer menu are described in the followi
67. 15 Enter the name of your experiment as the setup name then click Link amp Save Single Stained Setup Compensation calculation has completed successfully Name 4 Color experiment Link amp Save Apply Only Cancel Vv Tip To keep track of compensation setups include the experiment name date or both in the setup name NOTE We recommend 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 Chapter 4 Running Samples 137 Data Recording and Analysis Once you have optimized the cytometer electronics for your sample type you are ready to record and analyze data Before you record data we recommend that you gate out doublets in order to record only singlet events The Doublet Discrimination Gating template provides gated plots for this purpose During analysis recorded data is displayed in plots and gates are used to define populations of interest BD FACSDiva software analyzes the gated data and calculates statistics that you can print or export With global worksheets data can be shown for a series of tubes on the same worksheet manually or in an automated batch analysis The following sections describe how to use BD FACSDiva software features to record and analyze sample data 138 BD FACSAria II User s Guide Setting Up the Experiment Before you record data set up an experi
68. 4 Way Purity mode or select a mode with a yield mask of zero For more information see Yield Mask on page 54 1 Create a new sort layout by clicking the New Sort Layout button on the Browser toolbar By default the 2 Tube Sort Layout appears 2 Make appropriate entries in the Sort Layout window amp Tube 001 Sort Layout _001 Device Precision Target Events Save Sort Reports Save Conflicts Sen 2 Tube Purity v Ask User x oO Left Right A Sort Rate Confl Cnt Confl Rate Efficiency View Counters e Select the collection device from the Device menu e Change the sort precision mode to Purity two tubes 4 Way Purity four tubes or Single Cell plate or slide e Enter the number of target events by selecting a value from the menu or entering a number in the field e Select a Save Sort Reports option Save None Save All or Ask User See Setting Up a Sort Layout on page 89 e Select the Save Conflicts checkbox if you are using a 2 or 4 Tube Sort Layout and want to save conflicts 162 BD FACSAria II User s Guide 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 Add the required population s to each sort location field To display fewer counters in the Sort Layout window click the View Counters button and deselect a menu option The c
69. 5 C and 15 psig for 30 minutes with a 7 5 minute warmup and shutdown cycle Fill the sterilized sheath tank with sterile sheath fluid Obtain a new sheath filter to replace the old filter when instructed to do so in the wizard See Changing the Sheath Filter on page 192 Install the decontaminated sheath probe into the sheath tank and tighten the nut securely with an 11 16 inch wrench Install the top section of the containment device into the bottom section and then tighten the thumbscrew Chapter 6 Shutdown and Maintenance 187 9 Sterilize the DI water sensor by soaking in a 10 bleach solution for 10 minutes 10 Fill the DI water container with sterile DI water and 3 mL of bleach per liter of DI water and then reinstall the sterilized DI water sensor To run the Prepare for Aseptic Sort command 1 Select Cytometer gt Cleaning Modes gt Prepare for Aseptic Sort Follow the instructions on screen as you perform the procedure Aseptic Sort Prep This procedure takes approximately 45 minutes to complete and requires the following 1 Sterilized sheath tank with sterile sheath 1 2 Sterilized Dl water sensor and tank filled with sterile water 3 New 0 2 um sheath filter Please install these components as instructed during this procedure 68 Ensure that the closed loop nozzle is in place 69 Connect the bleach tank line to the DI water port 2 Install the integrated closed loop nozzle in the flow cell and click D
70. 5 000 Setting Up the Global Worksheet A global worksheet is used to perform doublet discrimination and to set up plots to preview and record data 1 Click the Worksheets View button on the Worksheet toolbar The global worksheet is shown If you are using the Doublet Discrimination Gating template the worksheet shows the predefined plots and gates used to distinguish singlets from doublets The second and third plots are set up to display gated data from the first and second plots respectively as shown in the population hierarchy 2 Create two dot plots for previewing and recording data For this example create a FITC vs PE plot and an APC vs PerCP Cy5 5 plot Mi Tip Double click the Plot button to keep it selected until you create all plots Click any other button to undo the selection 140 BD FACSAria II User s Guide Turn on biexponential display a Select the two plots b In the Inspector select the checkboxes for X Axis and Y Axis under Biexponential Display See Figure 4 25 Figure 4 25 Turning on biexponential display Inspector Dot Plot Plot Title Labels Acquisition Dot Plot Tube iment Analysis Sample 4 color_001 X Parameter FITC 4 v Y Parameter PE A v Biexponential Display X Axis Plot Elements C Grid Background Color V Grid Outline V Tick Marks E Ful Half Plot Outline V Tick Labels M M
71. 5 Removing the FSC ND filter Nozzle holder FSC ND filter Set screw 3 To reinstall the filter slide it into the slot with the filter side down and the label facing the flow cell NOTE You can remove the nozzle holder to get better access to the ND filter slot 224 BD FACSAria II 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 Reference Manual If additional assistance is required contact your local BD Biosciences technical support representative See Technical Assistance on page xv Troubleshooting suggestions in this chapter are grouped under the following headings Troubleshooting the Stream on page 226 Troubleshooting the Breakoff on page 231 Sorting Troubleshooting on page 232 Acquisition Troubleshooting on page 237 Fluidics Troubleshooting on page 244 Electronics Troubleshooting on page 245 225 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 screws See Figure 1 12 on page 14 Nozzle inserted imp
72. 75 nm laser Sensitivity enables separation of fixed platelets from noise identification of bacteria and 0 5 micron beads Sample acquisition rate Maximum acquisition rate 70 000 events per second with 8 parameters 12 compensation pairs Maximum rate lt 70 000 events per second with more parameters gt 100 000 events per second with fewer parameters Chapter 8 Technical Specifications 249 Sort Performance Table 8 4 Sort performance specifications Drop drive frequency Range from 1 to 100 000 Hz Purity and yield At 70 psi and 90 kHz with an average threshold rate of 25 000 events per second for a four way sort results in a purity of gt 98 and a yield gt 80 of Poisson s expected yield Viability Sorts were performed at a variety of sheath pressures using cell lines and human peripheral blood mononuclear cells All sorts resulted in cells that proved viable and proliferated for several days post sort Sort collection devices Two way sorting into the following sample tube collection devices 12 x 75 mm tubes and 15 mL tubes Four way sorting into the following sample tube collection devices microtubes 12 x 75 mm tubes and 1 5 mL Eppendorf tubes Automatic Cell Deposition Unit ACDU option allows for slide and plate sorting into 6 24 48 96 and 384 well plates Sample collection cooling Water recirculator for refrigeration heating optional BD FACS Ac
73. 98 515 3 3 100 022 5 3 101 079 36 97 146 36 eS 5 Be be a kE 3 es mT LI MAASI ASAE s 100 i50 200 250 so 10 150 200 250 PerCP Cy5 5 A amp 1 000 PE Cy7 A 1 000 Record Date Apr 24 2006 2 28 21 PM SOP Administrator Serial 22300049 Comments Changed sheath fitter ran long clean FSC A FSC A SSC A SSC A FITC A FITC A PE A PE A Population Events Mean cv Mean cv Mean cv Mean cv Bei 8536 98 091 32 100 852 22 100 029 34 99 541 34 PE Texes Red A PE TexasRed A PerCP Cy5 5 A PerCP Cy5 5 A PE Cy7 A PE Cy7 A Population Mean cy Mean cy Mean cy Er 98 171 34 98 974 38 99 271 64 321 Reusing the QC Experiment The QC experiment was designed so you can reuse it for subsequent QC runs by duplicating the tube without data Collecting all QC data in the same experiment makes it easy to export the statistics to a QC log 1 Verify that the cytometer configuration is set to the appropriate configuration for QC 2 Open the QC experiment 3 Right click the most recent tube and select Duplicate without Data 4 Rename the tube with the current date 5 Verify that area scaling and laser delay are correct a Verify that the Area Scaling worksheet is displayed b Prepare and load a tube with QC particles c Verify that the FSC and blue laser area scaling factors are set correctly Vv Tip You can use the same area scaling values that were determined the last time you ran the QC particles Select the last recorded data file in your QC experime
74. BD FACSAria II cytometer uses innovative designs for both the excitation and collection optics The optics can be viewed by opening the optics access door and the flow cell access door A 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 achromatic focusing lens as shown in Figure 1 17 on page 20 For information about how signals are generated see Signal Generation on page 32 18 BD FACSAria II User s Guide The BD FACSAria II cytometer uses low powered air cooled and solid state lasers that do not have special power or cooling requirements See Table 1 2 for the laser choices See Table 8 5 on page 251 for laser specifications Table 1 2 Laser configurations Laser Wavelength Comments Blue 488 nm Standard Red 633 nm Standard Violet 405 nm Optional Near UV 375 nm Optional Uses the same optical path and PMTs as violet laser See Appendix B on page 265 a When both the Near UV and violet lasers are installed only one of them can be used at the same time The laser power toggle switch determines which aser is active A BX ticks emit intense coherent electromagnetic radiation that can cause irreparable damage to human sk
75. D filter For applications involving large particles where events appear off scale on the FSC axis with a gain of zero a higher value ND filter is needed to decrease the FSC signal and keep the events on scale The system comes with three ND filters 1 1 5 and 2 See Removing or Installing the FSC ND Filter on page 223 Chapter 2 Theory of Operation 39 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 II flow cytometer the photodiode and photomultiplier tubes PMTs The photodiode is less sensitive to light signals than the PMTs and 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 FACSDiva software the Cytometer Configuration window 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 select the appropriate fluorochrome within your software experiment See Cytometer Configuration on page 68 for more information
76. NOTE You can also use the quality control process originally established in previous versions of BD FACSDiva software See QC Using BD FACSDiva Software on page 307 To run a performance check start the Cytometer Setup and Tracking CS amp T application within BD FACSDiva software and select the appropriate cytometer configuration Each configuration must have a valid baseline See the BD Cytometer Setup and Tracking Application Guide for more information on running a baseline BD FACSAria II User s Guide Preparing the CS amp T Workspace To ensure that your cytometer is performing consistently over time it s important to keep as many variables constant as possible such as bead type sheath pressure and cytometer settings For this reason you should run the daily performance check using the same cytometer configuration each day 1 Turn off the Sweet Spot if it is on then select Cytometer gt CST The cytometer disconnects from the BD FACSDiva interface and connects to the Cytometer Setup and Tracking interface A window similar to Figure 4 12 on page 117 opens Figure 4 12 Main CS amp T window Cytometer Setup and Tracking Gie Cytometer Tools Setup Reports Performance Tracking System Summary OK Cytometer Canhguraton 130 20 Violet PerCP Cy5 5 AmCyan Lot ID 85631 E Cytorrete Baseine October 23 2007 03 13 AM EA Cytomater Perfomance October 23 2007 08 16 AM E Cytomete Pedomance Rezutx Passed DER
77. O Ring The O ring at the bottom of the sample injection chamber should be lubricated as needed to maintain proper operation 1 Verify that the loading port is in the down position and open the hinged cover 2 Check the O ring located at the opening in the bottom of the chamber to see if it is dry 220 BD FACSAria II User s Guide If the O ring is dry it must be lubricated with O ring lubricant from the accessory kit a Remove the clear plastic tube holder from the tube holder base b Apply a small amount of O ring lubricant to the O ring inside the chamber and to the top outside beveled edge of the tube holder base This is where the base contacts the O ring inside the sample injection chamber c Wipe off any excess lubricant d Replace the tube holder on the base Figure 6 34 Loading port with tube holder removed Apply lubricant to outside beveled edge of tube holder base Chapter 6 Shutdown and Maintenance 221 222 Using Custom Optical Filters If you want to install a custom filter or dichroic the filter should comply with the following specifications Table 6 4 BD FACSAria II 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 the center of the octagon or trigon shoul
78. Population s sort count and target event count for each sort location field Chapter 3 Using BD FACSDiva Software 95 96 Figure 3 14 Typical sort report xperiment Aseptic 85 Specimen Specimen_001 ube Sort Sort Layout Sort Layout_002 Application FACSDiva Version 6 1 Sort Settings Sort Setup Frequency Amplitude Phase Drop Delay Attenuation Precision Yield Mask Purity Mask Side Stream Voltage Far Left 81 00 Neighboring Drop Charge 2nd 14 00 Acquisition Counters Threshold Count Processed Events Count evt Electronic Aborts Count evt Sort Elapsed Time hh mmiss Sort Counters Sort Rate evt s Conflicts Count evt Conflicts Rate evt s Efficiency Sort Layout Sort Report 85 micron 47 0 43 6 0 00 25 58 off 4 Way Purity 0 32 Left 28 00 3rd 3 00 Far Left Left 1786 288 235757 50869 285 61 86 82 Far Left Left CD4 1477612 CD19 238969 Report Date 2007 09 12 at 15 47 06 Device 4 Tube User ID Jane Cytometer Pilot 2 FACSAriall P94400001 Phase Mask 0 Single Cell off Sweet Spot On First Drop 105 Target Gap 7 Plates Voltage 5 000 voltage Centering 0 Sheath Pressure 45 00 Right Far Right 27 00 72 00 4th 0 00 4882994 4900425 95344 00 13 47 Right Far Right 520 1074 121104 151070 146 182 78 85 Right Far Right CD16 56 A 430693 CD8 888900 The Sort Report window contains a File menu where you can select to print or export the report E
79. Research Use Only It is not for use in diagnostic or therapeutic procedures Using the BD Temperature Control Option Note that before you start the recirculating water bath you must attach the tubing to the appropriate collection device Setting Up the Water Bath 1 2 Remove the threaded plug from the output port on the water bath Ensure that the drain cock on the back of the water bath is closed by turning it fully clockwise Set the pump outflow to maximum by turning the knob fully counter clockwise Remove the top cover to access the knob which is located inside the water bath towards the back See the operating instructions supplied with water bath for additional details on this process This is referred to as position 1 Connect the clear tubing end of the insulated hoses to the input and output ports on the water bath See Figure D 2 on page 298 Slide the tubing over the hose barbs and twist gently while installing to get the tubing completely over the barbs Appendix D Temperature Control Option 297 Figure D 2 Input and output ports on water bath 5 Connect the insulated hoses from the recirculating water bath to the cytometer control panel NOTE Make sure you match the flow direction from the water bath port to the flow direction on the instrument manifold The instrument ports are labeled cooling in and cooling out Figure D 3 Connections for temperature control option Coolingin Cooling o
80. Right B Fic 17752 J erc 20910 Far Right I Fercr cys 18389 Sort Rate Confl Cnt Confl Rate Efficiency 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 stream charging wire attached to the flow cell See Figure 2 17 on page 53 BD FACSAria II User s Guide Figure 2 17 Flow cell with stream charging wire Stream charging wire 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
81. Screw the nuts with connecting tubing onto each end of the filter and tighten finger tight b Tighten nuts 1 to 2 additional turns with a wrench c Check for leaks once pressure is turned on d Write the current date on the filter so you will know when to replace it Open the bleeder valve on top of the filter a small amount and leave it open until fluid seeps out through the valve Close the valve Changing the Ethanol Shutdown Filter We recommend changing the ethanol shutdown filter every 6 months Spare filters are included with the accessory kit NOTE The ethanol shutdown filter is the same as the one used for the sheath filter The filters can be used for either purpose 1 Remove the filter by pressing the tabs on each quick disconnect coupling See Figure 6 11 Figure 6 11 Ethanol filter Direction of flow gt mp p gt Unscrew the nuts at each end of the filter to remove the connecting tubing Locate a new filter and wrap the threads at each end with Teflon tape Chapter 6 Shutdown and Maintenance 193 4 Observe the direction of flow on the filter and replace it in the same orientation a Screw the nuts with connecting tubing onto each end of the filter and tighten finger tight b Tighten nuts 1 to 2 additional turns with a wrench c Check for leaks once pressure is turned on d Write the current date on the filter so you will know when to replace it 5 Ope
82. See Setting Up a Sort Layout on page 89 Click Stop Acquiring in the Acquisition Dashboard to stop the sample flow 166 BD FACSAria II User s Guide 10 Turn off the deflection plates by clicking the Voltage button in the Side Stream window Remove the lower section of collection tube holder by lifting up on the handle and pulling the lower section of the holder down and forward Replace collection tubes as needed Reinstall the tube holder and pull down on the handle to secure it in place Click Acquire Data in the Acquisition Dashboard to restart the sample flow Click the Resume button in the Sort Layout window to continue sorting Click OK when you are prompted to open the aspirator drawer or turn on the deflection plates The sort counters resume from the value where they stopped The threshold counter restarts however the value is accumulated and the total count is saved in the final sort report Pausing and Resuming a Sort The Pause Resume feature allows you to temporarily pause the sort and still retain the sort counter values This is useful when you need to make adjustments to an experiment during a sort Be aware that the sample continues to flow during a pause NOTE If you need to replace the sample tube to refill it or to replace collection tubes you should stop the sort See Stopping and Resuming a Sort on page 166 1 To pause a sort while it is running click the Pause button in the Sort Layout window
83. See the BD Cytometer Setup and Tracking Application Guide for complete information on using CS amp T e Performance Tracking LJ Opens the performance tracking feature within the main CS amp T window See the BD Cytometer Setup and Tracking Application Guide for complete information on using this feature BD FACSAria II User s Guide Cytometer Configuration Window The BD FACSAria II cytometer is equipped with a specific set of lasers filters and dichroic mirrors The Cytometer Configuration window lets you define which fluorochromes or cell parameters will be measured at each photomultiplier tube PMT detector Within this window you can define parameters for an unlimited number of fluorochromes up to four lasers and all of the detectors Select Cytometer gt View Configurations to open the window shown in Figure 3 5 on page 70 Click the Parameters tabs for a list of parameters Click the Filters and Mirrors tab for the list of optics The Cytometer Configuration window also displays these settings e Sheath pressure e Nozzle size e Window extension See the BD Cytometer Setup and Tracking Application Guide for more information on editing configurations including adding parameters mirrors and filters Chapter 3 Using BD FACSDiva Software 69 Figure 3 5 Example configuration for a three laser system Cytometer Configuration Cytometer BD FACSAria Current Configuration 3 laser 9 color 5 2 2 Configuration 5 BD d
84. Select File gt Print Keep a record of the daily QC results for future reference By comparing results from day to day you will be able to monitor instrument performance 320 BD FACSAria II User s Guide An example of recorded data is shown in Figure E 6 Figure E 6 Recorded QC data Instrument QC Blue Laser Instrument QC Red and Violet Lasers Appendix E OC Using BD FACSDiva Software Rainbow 042406 Rainbow 042406 Rainbow 042406 Rainbow 042406 Rainbow 042406 g3 g Ee Eg 3 a k g zj z gi lt 3 Ee z Es 3 sey Be 38 34 33 Bo 8 8 87 GSE 4 af E s amp j j 33 ot ASALAIA MASAJ LAASI ALAJ A so 100 180 200 250 So 10 150 200 250 s 100 150 200 250 s 0 180 200 250 so 100 10 200 250 FSC A 1 000 FSC A 1 000 SSc A 1 000 APC A 1 000 APC Cy A 1 000 Rainbow 042406 Rainbow 042406 Rainbow 042406 Rainbow 042406 Rainbow 042406 x a x a a z5 2 z3 EL z4 3 am 3 35 37 amp 8 5 ESI Ez ES g i Ez AAAI LEAS LASAS LAASI LAASI ot ens 50 100 150 200 250 0 100 150 200 250 60 100 150 200 250 wo 150 200 20 o 150 200 250 FITC A 1 000 PEA 1 000 PE Texas Red A 1 000 DAPA amp 1 000 Alexa 430 A amp 1 000 g Rainbow 042408 Rainbow 042406 Bs 7 APC A APC A APC Cy7 A APC Cy7 A DAPA DAPIA Alexa 430 A Alexa 430 4 FE j Population Mean Cv Mean cv Mean cv Mean cv Pe
85. Setup gt Rainbow Beads High Sort Setup 8 gJ 24 gJ SJ zg ey SRJ 274 5 s4 og ai 3J sq E Bee T a py Ler neer 50 100 150 200 250 50 100 150 200 FITC A x 1 000 FITC A amp 1 000 Experiment Name Area Scaling Template Experiment Name Area Scaling Template Specimen Name Rainbow Beads Specimen Name Rainbow Beads Tube Name High Sort Setup Tube Name High Sort Setup Record Date Record Date SOP SOP FITC A FITC H FITC A FITC H Population Mean Mean Population Mean Mean Mri 69 530 100 072 ri 101 575 100 688 Adjusting Area Scaling and Laser Delay for the Red and Violet Lasers The laser delay factor in BD FACSDiva software is used to realign the signals so they can be measured and displayed on the same time scale Signals are aligned with respect to the blue laser so the blue laser will have a 0 delay value the red laser will have a negative delay value and the violet or Near UV laser will have a positive delay value 1 Adjust the APC voltage to place the APC H signal at approximately 100 x 10 The red laser area scaling and laser delay can be adjusted using any parameter excited by the red laser 2 Adjust the delay setting for the red laser if needed Appendix E OC Using BD FACSDiva Software 317 The sort setup enters area scaling and laser delay values from the last time the sort setup was used For this reason the settings should be close to the appropriate values You are simply confirming that the val
86. Solutions Problems using Auto Stream is not stable Make sure stream is stable before Delay feature starting to run auto delay Diode laser does not fully Adjust the diode laser to intercept intercept the sorting streams streams in the middle producing biggest and brightest spots in the left and center images Event rate is too low or too Adjust flow rate to increase or high decrease event rate 230 BD FACSAria II User s Guide Troubleshooting the Breakoff Use the following examples to help troubleshoot problems with the breakoff image Lia 70 micron x FA 70 micron x Stream Z Sweet Spot FA 70 micron x Stream Bl Sweet Spot FA 70 micron x Stream Z Sweet Spot FA 70 micron E Stream Z Sweet Spot FA 70 micron x Stream Z Sweet Spot i o O O E si B 9 a E E 2 f lt 6 2 6 306 163 elz Normal stream Nozzle Nozzle inserted Partial clog Wet or dirty Attenuation is image 4 inserted improperly or strobe lens on at wrong A amp improperly orifice is off pressure ao center 5 Remove the Remove the Remove the Clean the lens Turn off nozzle and nozzle and nozzle clean as described in attenuation in S a re insert it re insert it it and then Cleaning the the Side 5 x re insert it Camera Stream os Windows on window aa page 217 Chapter 7 Tr
87. Supply To connect the fluidics cart to an external air source switch on the Auxiliary Air Supply and attach the external air line to the air input connector The external air supply should provide 80 100 psi The external air must be dust and oil free Figure 1 4 Connectors on fluidics cart External air External air input connector supply switch Air pressure Circuit breaker gauge Voltage selector switch 4 BD FACSAria II User s Guide NOTE There is a pull out drip tray under the connection area Check the tray periodically for moisture See Checking the Fluidics Cart Drip Tray on page 203 for details Power and Operation Power to the fluidics cart is supplied by the cytometer The cart is activated when the cytometer main power switch is turned on see Power Panel on page 24 A A 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 cytometer Figure 1 3 on page 4 Do not plug the power cord directly into a wall socket Do not change the input voltage selection switch on the fluidics cart 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 fluidics cart is
88. The ULPA filter used in the BD AMO captures and retains 99 9995 of all particles down to and including particles 0 12 microns in size according to the manufacturer s specifications Figure C 2 ULPA filter N T Appendix C BD Aerosol Management Option 277 Operating the BD Aerosol Management Option Starting Up the Evacuator Before starting up the evacuator make sure that the ULPA filter is completely seated against the bottom of the evacuator filter well and that the tubing is securely attached to the filter and instrument manifold AA Any instrument surface that comes in contact with biological specimens can transmit potentially fatal disease Use universal precautions when handling instrument hardware Wear suitable protective clothing and gloves To start up the evacuator 1 Install the splash shield or the tube holder below the aspirator drawer Figure C 3 on page 279 The splash shield is required for sorting into a multiwell plate or onto a slide To install the splash shield a 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 b Remove the tube holder if one is installed c Insert the splash shield into the slotted fittings below the sort aspirator drawer See Figure C 3 on page 279 Push the splash shield all the way in 278 BD FACSAria II User s Guide 3 Figure C 3 Installing
89. Tubing on the Closed Loop Nozzle To replace the tubing on the closed loop nozzle use this procedure See Figure 6 23 Use a 7 inch length of sample tubing from the accessory kit as the replacement Figure 6 23 Replacing tubing from closed loop nozzle Ferrule Nut Tubing 7 inch length i Closed loop nozzle Union fitting To replace the tubing 1 Unscrew the nut from the closed loop nozzle and from the union fitting and pull out the tubing from both places Make sure the ferrule comes out on the tubing on both ends If not use the ferrule tool to remove it See Figure 6 15 on page 198 2 Slide the nut and ferrule off each end of the tubing 3 Slide the nut and ferrule onto the new tubing as shown in Figure 6 23 Insert the tubing into the closed loop nozzle and slowly tighten the nut until secure Do not over tighten Make sure the tubing is pushed all the way in while tightening the nut 4 Insert the tubing into the union fitting and slowly tighten the nut until secure Do not over tighten Pull gently on the tubing to ensure it is secure Chapter 6 Shutdown and Maintenance 211 Installing or Removing a Sample Line Filter Sample filters can be installed on the end of the sample line to filter out large particles from a sample Pre filtering the sample before beginning any sorting is recommended Two sizes of sample line filters are included in the accessories kit 35 micron green and 50 micron blue The fi
90. Violet or Near UV Red laser laser signal signal Octagon At installation the octagon and trigon arrays are set up with the filter and mirror combinations shown in Table 8 7 on page 252 You can rearrange these configurations according to the type of fluorochromes in your experiment BD FACSAria II User s Guide Stream Viewing Optics The BD FACSAria II cytometer is equipped with optical components that are used to view the stream Figure 1 21 e The upper camera generates 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 FACS Accudrop option It enhances the ability to see side streams and assists in setting an accurate drop delay value Figure 1 21 Stream viewing optics Diode laser Micrometer dial Lower camera viewing window 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 44 for more information about viewing the streams and to learn how Accudrop components are used to determine the drop delay Chapter 1 Cytometer Components 23 24 Cytometer Electronics The electronic components consist of power controls and connectors along with processing boards in the card cage This section describes only user adjustable cytometer electronics For more information about the electr
91. acement parts Item Part No Autoclavable 10 L container 340261 Auxiliary 5 L container 333504 Sheath sensor probe 642012 Waste sensor probe 6 level 334915 Auxiliary sensor probe non ethanol 343835 Auxiliary sensor probe ethanol 642874 Air filter for cabinet side door 334351 Air filter for ACDU cabinet set of 3 334821 ULPA filter and tubing replacement kit 334822 set of 3 for use with the AMO Four way 1 mL collection tube holder 641614 260 BD FACSAria II User s Guide Consumables Cytometer Setup Particles Particle Supplier Catalog No BD Calibrite beads BD Biosciences e Two color kit 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 1m beads Polysciences Inc 18604 for the 405 nm laser 800 523 2575 SPHERO Rainbow Calibration BD Biosciences e 559123 Particles 3 0 3 4 um 8 peaks e 556286 e 556291 brightest peak in 556286 BD FACS Accudrop beads BD Biosciences 345249 BD Cytometer Setup and Tracking BD Biosciences 641319 CS amp T beads 1 vial 642412 3 vials Appendix A Supplies and Consumables 261 Reagents Reagent Supplier Catalog No BD FACSFlow sheath fluid BD Biosciences 340398 US and Latin America 342003 other countri
92. add a mirror click Add under the Mirror list b Select a pass type and enter a wavelength in the field provided Chapter 3 Using BD FACSDiva Software 75 76 Filters Mirrors Band Pass 585 15 Long Pass 575 BandPass 576 26 Long Pass 556 Band Pass 575 26 Long Pass 550 BandPass 560 20 LongPass 545 BandPass 530 30 LongPass 505 Band Pass 525 50 Long Pass 502 Band Pass 510 50 Long Pass 475 Band Pass 488 10 Band Pass 485 22 Band Pass 450 50 Band Pass 450 40 Band Pass 450 20 Band Pass 440 40 Band Pass 405 20 vv Copying a Base Configuration You cannot edit or delete your base configuration However you can use it as a starting point to create a custom configuration 1 In the Configurations tab right click the Base Configurations folder and select New Folder 2 Rename the new folder Custom Configurations 3 Right click the base configuration and select Copy 4 Right click the Custom Configurations folder and select Paste 5 A copy of the base configuration is added to the Custom Configurations folder Configurations Parameters Filters and Mirrors Base Configurations E 3 laser 9 color 5 2 2 Configuration 5 01 02 07 E amp Custom Configurations E Copy OF 3 laser 9 color Configuration 5 105 08 07 ai BD FACSAria II User s Guide 6 Enter a descriptive name and press Enter For example use the name 70 70 meaning 70 micron nozzle at 70 psi If y
93. age 178 for more details Change Sample Filter Sample line filters can be installed to filter a sample before sorting This selection opens the Sample Filter Change wizard which guides you through the process See Installing or Removing a Sample Line Filter on page 212 for the instructions Cleaning Modes BD FACSDiva software contains pre programmed cleaning modes that are activated by selecting the corresponding menu command from the Cytometer gt Cleaning Modes menu See Internal Cleaning on page 183 for more information Fluidics Startup Fluidics Shutdown Change Sample Filter Cleaning Modes Sample Line Backflush Sheath Pressure Clean Flow Cell Sample Agitation gt Prime after Tank Refill Sample Temperature gt Prepare for Aseptic Sort Cytometer Details View Configurations CST Performance Tracking LJ Catalogs Standby Sheath Pressure The sheath pressure determines how quickly particles pass through the laser beam Select Cytometer gt Sheath Pressure to view the current sheath pressure and change the pressure for custom sort setups BD FACSAria II User s Guide F Sheath Pressure Sheath Pressure Adjustment Sheath Pressure a St A Each sort setup option is optimized at a preset sheath pressure If you change the sheath pressure many other values will be affected and need updating including the drop drive frequency drop charge levels laser delay and area scaling
94. age 51 There are two methods for determining the drop delay e Manual drop delay Using the standard method as described in the following sections e Auto drop delay Using an automated algorithm method See Determining the Drop Delay Automatic Method on page 159 154 BD FACSAria II User s Guide NOTE Before beginning these procedures 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 the drop delay using the Accudrop experiment template Because no data is recorded the experiment can be reused as often as you like 2 3 Create an experiment from the Accudrop Drop Delay template Select Experiment gt New Experiment Select the Accudrop Drop Delay experiment and click OK Experiment Templates General G5 Practice a Prana Name Accudrap Drop Delay Blank Experiment Accudrop Drop Delay 11 22 06 11 00 AM Blank Experiment with Sample Tube 11 22 06 11 00 AM jDoublet Discrimination Gating 11 22 06 11 00 AM QC Experiment 11 22 06 11 00 4M i Name Accudrop Drop Delay jes p Drop Delay Copies 1 St Expand Specimen_001 and Tube_001 Set the current tube pointer to Tube_001 Chapter 5 Sorting 155 Open the sort layout by double clicking it LL Accudrop Drop Delay 8S Cytometer Settings 8 EN Specimen_001 G U Tube_oo1 k Analysis Sort Layout_O001 Using Manual Drop
95. always default to off For more information see Sort Setup on page 85 Chapter 2 Theory of Operation 49 50 Table 2 2 Side Stream window controls Control Description Voltage button On Off Turns the plate voltage on and off Test Sort button gr FA Off On Generates test side streams based on test sort pulses Optical Filter button Y F In Out Controls the position of the optical filter in front of the lower Accudrop camera Attenuation button Aj w off On Decreases the amplitude of the drop drive At lower pressures you may need to turn on attenuation to dampen the amplitude Waste Drawer button da nu Ue Closed Open Opens or closes the aspirator drawer depending on its current state The default state is closed For more information see Aspirator Drawer on page 15 Drop Delay field 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 Auto Delay Opens the Auto Drop Delay dialog Voltage sliders far left left right far right Set the percentage of charge to be applied to the corresponding stream as a percentage of maximum Voltage Center slider Adjusts the relative voltage between the left and right plates which moves the streams slightly to the le
96. and spectral grade methanol or absolute ethanol in a dropper bottle do not use acetone to clean the optical filters Wrap a triangular section of the lens paper around the cotton end of a cotton swab Moisten and seal the end with a few drops of alcohol Holding the cotton swab in a horizontal position gently rub any spots on the filter surface and wipe clean Allow the solvent to evaporate and check the filter surface for streaks Inspect a 4 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 Removing or Installing the FSC ND Filter For applications involving large particles where events appear off scale on the FSC axis with a voltage of zero keep the FSC ND filter in place to decrease the FSC signal and keep the events on scale For applications involving small particles for example bacteria or platelets you might need to remove the FSC ND filter as follows 1 2 Open the flow cell access door Locate and pull out the FSC ND filter Figure 6 35 on page 224 Chapter 6 Shutdown and Maintenance 223 The filter is installed at the left end of the FSC detector block just to the right of the flow cell To remove the filter loosen the set screw and pull the filter out of the slot Figure 6 3
97. and the chamber pressure forces sample through the sample line into the cuvette flow cell To stop sample flow after a tube is loaded click the Stop Acquiring button The sample flow rate is specified using the Flow Rate field in the Acquisition Dashboard You can adjust the flow rate from 1 11 which corresponds to approximately 10 80 uL min Figure 2 4 Load button and Flow Rate field in Acquisition Dashboard Acquisition Dashboard Current Activity Active TubeWell Threshold Rate Stopping Gate Events Elapsed Time Tube 001 0 evtis Oevt 00 00 00 Basic Controls aw mano Mish e Acquistion Setup Stopping Gate E All Events v Events To Record 10000 evt v Stopping Time sec 0 Sf Storage Gate All Events wv Events To Display 1000 evt Flow Rate 1 0 SH Acquistion Status Processed Events Electronic Abort Rate Threshold Count Electronic Abort Count Note that the relatively longer sample tubing on the BD FACSAria II cytometer results in a different flow rate between cells and beads Thus absolute counting using BD Trucount beads can yield erroneous results BD FACSAria II User s Guide 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 flui
98. asers 177 181 side populations 272 side scatter SSC 32 side streams optimizing 170 window 49 84 signals about 32 detection 34 fluorescent 33 generating 41 low Area 239 no fluorescent 238 scattered light 32 troubleshooting 243 Single Cell mode 58 slides installing 169 302 sorting into 168 software about 26 cleaning modes 183 components 62 83 cytometer controls 63 QC 308 templates 97 sort block 13 16 collection chamber 17 devices 17 Sort button 92 sort layouts about 52 87 creating 89 162 custom 84 172 editing 91 entering populations 90 162 sort menu 84 sort precision modes 4 Way Purity 57 162 about 53 Index 335 creating 59 84 defaults 57 deleting 59 Fine Tune 58 Initial 58 Purity 57 Single Cell 58 Yield 58 sort reports about 94 exporting 96 165 printing 96 165 sort setup values 85 86 sort test 50 153 sorting about 44 149 Accudrop beads 158 analysis 143 151 161 aseptic 187 aspirator drawer control 50 92 bulk 152 collection devices 87 conflicts 53 91 93 162 235 controls 83 92 counters 93 experiment 162 gates 161 into plates 168 into slides 168 into tubes 152 monitoring 94 165 pausing 46 92 167 populations 52 90 161 162 recording data 165 resuming 166 167 setup 150 starting 92 164 stopping 92 troubleshooting 232 336 BD FACSAria II User s Guide specifications cytometer 248 fluidics cart 254 laser 251 temperature control option 305 splash shield ins
99. atio tab views Data already recorded Create a new tube Chapter 7 Troubleshooting 243 Fluidics Troubleshooting Observation No fluid in line during system prime Possible Causes Air lock in sheath or fluidics 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 Prepare for aseptic sort fails Air lock in filter See previous recommendations Fluid line is detached Verify the fluid line connections on the fluidics cart and on the cytometer Push firmly on each line to ensure it is connected Fluidics cart air flow lt 80 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 wipe up any water daily Bleeder valve is open Check and close all bleeder valves for fluid and sheath filters Broken fluid line Contact BD Biosciences
100. ation on the nozzle e Changing the Integrated Nozzle on page 205 e Cleaning the Integrated Nozzle on page 206 BD FACSAria II User s Guide Table 1 1 Nozzle handling recommendations Recommended Action Result Always use the integrated closed loop nozzle Keeps the flow cell clean and reduces for cleaning and shutdown procedures chances for clogs A clean flow cell provides improved sensitivity and higher performance Do not expose integrated nozzles to bleach or Prevents the seal from coming loose detergents such as CONTRAD 70 Avoid and falling out any strong base solutions Do not wipe the surface of the seal with Prevents damage to the seal that could anything result in leaking If the seal in an integrated nozzle eventually comes out or gets damaged you can replace the seal with a standard O ring See Temporary Replacement of a Seal on page 208 Standard Nozzle Early BD FACSAria II systems were shipped with standard nozzles with a replaceable O ring If your system is equipped with standard nozzles see Using the Standard Nozzle on page 208 for details on handling and maintaining them 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 draw
101. atures a 375 nm solid state laser that enables sorting and analysis of side populations This Near UV laser uses the same emission pathways as the violet laser option and the laser alignment is fixed and requires no adjustments once it is installed The Near UV laser can be added at the factory as an option on a new instrument or it can be installed by a BD Field Service Engineer into an existing system in the field Figure B 1 BD FACSAria II system with Near UV laser installed s J 375 nm Near UV laser 265 System Laser Configurations The BD FACSAria II systems can be configured as two laser three laser or four laser systems See Table B 1 for the system configurations See Figure B 2 for an illustration of the laser power toggle switch on a four laser system Table B 1 BD FACSAria II laser configurations System Configuration Blue Laser Red Laser Violet Laser alas 488 nm 633 nm 405 nm nm Two laser Yes Yes No No Three laser with Violet Yes Yes Yes No Three laser with Near UV Yes Yes No Yes Four laser Yes Yes Yes Yes NOTE Ina four laser system you can use either the violet or the Near UV laser but you cannot use them at the same time Figure B 2 Violet Near UV laser toggle switch 266 BD FACSAria II User s Guide Specifications Nominal Laser Wavelength nm Power mW Near UV laser 375 7 Safety Make sure that everyone wo
102. ay 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 cytometer 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 severe problem such as a clog is detected by the Sweet Spot the stream is shut off and sorting is stopped the deflection plates shut off the aspirator drawer closes and the sample tube is unloaded Pre programmed values can be downloaded to the Breakoff window by selecting one of the nozzle sizes 70 85 100 130 micron from the Sort gt Sort Setup menu Serre Target Actual value value BD FACSAria II User s Guide Note that changes to values in the Sort Setup windows Breakoff and Side Stream are automatically saved At startup the last s
103. bdbiosciences com Part No 644832 Revision A March 2009 BD FACSAria II User s Guide AY BD Biosciences San Jose CA 95131 1807 USA Tel 877 232 8995 Fax 800 325 9637 facservice bd com Asia Pacific Tel 65 6 861 0633 Fax 65 6 860 1593 Europe Tel 32 2 400 98 95 Fax 32 2 401 70 94 help biosciences europe bd com Brazil Toll Free 0800 771 7157 Tel 55 11 5185 9995 Fax 55 11 5185 9895 bdbiosciences brasil bd com Japan Nippon Becton Dickinson Company Ltd Toll Free 0120 8555 90 Tel 81 24 593 5405 Fax 81 24 593 5761 Canada Toll Free 888 259 0187 Tel 905 542 8028 Fax 888 229 9918 canada bd com Mexico Toll Free 01 800 236 2543 Tel 52 55 5999 8296 Fax 52 55 5999 8288 biocienciasmexico bd com 2009 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 n
104. ber door b Remove the tube holder if one is installed c Insert the splash shield into the slotted fittings below the sort aspirator drawer Push the splash shield all the way in 2 Click the Access Stage button to bring the ACDU stage to the front a Open an experiment if one is not already open and create a sort layout for any of the tubes b Inthe Sort Layout view click the Access Stage button to move the stage to the front of the sort collection chamber 3 Attach the recirculating water tubing to the ACDU stage Note that the tubing ports are labeled In and Out Attach the input tubing to the port on the left side of the stage and the output tubing to the port on the right side as shown in Figure D 5 on page 302 To attach the tubing push it into the port until the tubing snaps into place Appendix D Temperature Control Option 301 Figure D 5 Setting up the temperature control on ACDU stage Input tubing Output tubing If you need to remove the tubing push in the orange collar as you pull the tubing out of the port 4 Install the required collection device on the stage 5 Close the sort collection chamber door and start up the water bath 302 BD FACSAria II User s Guide Starting Up the Water Bath NOTE To ensure that the sample collection device is at the correct temperature start up the water bath at least 30 minutes 115 V model or 60 minutes 110 V model before you start sorting 1 Switch o
105. between when a particle intercepts the laser and when it reaches the droplet breakoff point is determined using BD FACS Accudrop technology see Drop Delay Overview on page 51 When a particle is detected and 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 14 on page 45 The following sections describe how the BD FACSAria II cytometer creates and charges drops how the drops are deflected and how sorting populations are identified BD FACSAria II User s Guide Figure 2 14 Sorting Charge is applied via the stream charging wire in the barb The sample generates light scatter and fluorescence signal The signal is analyzed The charged droplet breaks off Deflection plates attract or repel the charged droplet Uncharged droplets pass to waste Charged drops containing particles of interest are collected Oo ec Drop Formation
106. cal regulations Wear suitable protective clothing eyewear and gloves 5 Insert the new ULPA filter into the evacuator filter well Figure C 8 Figure C 8 Inserting the new filter 286 BD FACSAria II User s Guide 6 Push down on the filter to ensure that it is seated against the bottom of the filter chamber Figure C 9 on page 287 Figure C 9 Seating the new filter NOTE For optimal evacuation of aerosols the filter must be completely seated in the evacuator filter well 7 Liftthe spring loaded filter hold down and place it on top of the filter 8 Press and hold the Filter Life Reset button on the membrane panel for 5 10 seconds Reset button The green and amber LED lights will turn off and then on Hold down the button until the 100 indicator light is lit This resets the 180 hour filter life clock Appendix C BD Aerosol Management Option 287 9 Connect one end of the replacement tubing to the ULPA filter and the other end to the tubing manifold Figure C 10 on page 288 Figure C 10 Connecting new tubing NOTE For optimal evacuation of aerosols ensure that the tubing is securely connected at both ends 10 Connect the evacuator power plug to the power source Replacing the Air Filter To extend the life of your ULPA filter we recommend that you replace the air filter on a monthly basis The filter traps airborne dust that could clog the ULPA filter Regular replacement of the air filter will ext
107. ce eee nee 261 Reagents craact oeaiei a care ag See dower bea Surepe ga eee Saree dee 262 Labware ict cst ok Math a eek i et oto aera SRO Rae eos 263 Appendix B Near UV Laser Option 265 System Laser Configurations 00 cece cc cee cent en enns 266 Specifications secinu ea Ue eR a ea eee 267 Safety aam ect ash tees ees k Uy hacia ctelst a e bw hotels ob thal ty eagle ewe hi ei a 267 Operationy its sede E E AE E E Se iE Soe 3g te od Sok Cade ede 8 268 Selecting Optical Filters 2 2 2 eee eee ene 268 Creating a Custom Configuration 0 0 00 eee eee eens 269 Switching From the Violet to the Near UV Laser 0 0 eevee 270 Switching Back to the Violet Laser 0 0 cc cece eee eens 271 Side Population Application Guidelines 0 0 0 0 cece eee ene es 272 Instrument Setup si ee ee AAAS AAA SONAR A RER Ss 272 Experiment Setup Tips os Dere nae Saee ss Gs Galas alae Se ON 272 Troubleshooting 45 4 44 cove ek wee eee ete hy Pw ee a hie ede Ale bien 274 x BD FACSAria II User s Guide Appendix C BD Aerosol Management Option 275 Option Componer ove pus vd Ca epee tv eye HES Oy hue HES ews 276 EVaCuators inte Sty e piel Ae led Sah es Aad At E E El 276 ULPA Filter ads Bo boa sad Mob ela Bees Sees debs Bene OaS 277 Operating the BD Aerosol Management Option 00 ce eee 278 Starting Up the Evacuator 2 1 eee eee ene nen eens 278 Setting Up for Sorting oeae sod era ad fetid Peres Agel baa Pa RS 281 O
108. cell then click Done 102 BD FACSAria II User s Guide The fluidics startup process starts and the progress is displayed at the bottom of the dialog 638 Remove the closed loop nozzle from the flow cell assembly Fluidics Startup in progress Done 100 Turn the nozzle locking lever counter clockwise to the 6 00 position pull the closed loop nozzle out of the cuvette flow cell then click Done Caution Do not open the sort block door while the nozzle locking lever is in the 6 00 position The flow cell can be damaged by the top of the door during opening Always turn the lever clockwise back to the 12 00 position before opening the sort block door Insert the correct nozzle size in the flow cell a Verify that the seal in an integrated nozzle or the O ring in a standard nozzle is installed in the nozzle groove b Make sure the top side of the nozzle is facing up as you insert the nozzle c Push the nozzle all the way back into the flow cell d Turn the nozzle locking lever clockwise to the 12 00 position Click OK to complete the process When fluidics startup is complete select Sort gt Sort Setup and verify that the setup mode matches the nozzle size For information about Sort Setup modes see Sort Setup on page 85 Chapter 4 Running Samples 103 Starting the Stream The system can take anywhere from 20 seconds to several minutes to reach the correct pressure and turn on the stream d
109. ces is not liable for any claims related to or resulting from the buyer user s failure to install and maintain virus protection BD FACSAria II User s Guide Cytometer Components The BD FACSAria II flow cytometer is a high speed fixed alignment benchtop cell sorter The cytometer 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 II flow cytometer enables multicolor analysis of up to 13 fluorescent markers and two scatter parameters at a time The BD FACSAria II system consists of three major components a fluidics cart a benchtop flow cytometer and a workstation see Figure 1 1 on page 2 Nearly all cytometer functions are operated from within BD FACSDiva software For a description of the system components see the following sections For technical information about how the cytometer works see Chapter 2 e Fluidics Cart on page 2 e Flow Cytometer on page 6 e Workstation on page 26 Chapter 1 Cytometer Components 1 Figure 1 1 BD FACSAria Il cytometer components SBD FAcSArign u Fluidics Cart A separate fluidics cart supplies sheath and cleaning fluids and collects waste from the cytometer The self contained fluidics cart supplies the required air pressure and vacuum which eliminates the need for an external source although the cart can be hooked up to an in house air source if one is available The air pu
110. chamber 220 P parameters about 42 adding 40 69 74 labels 139 measuring 42 scatter distorted 241 parts replacement 260 pausing sorting 46 92 167 performance check running 120 tracking 68 phase field 51 masks 54 56 photodiodes 40 photomultiplier tubes PMTs about 22 40 applying voltages 42 assigning 40 69 74 optimizing for application settings 128 pinch valve changing tubing 214 collet fitting 216 plate loader 18 plate voltage 51 plates deflection 15 152 153 removing 219 installing 169 302 sorting into 168 plots excessive debris 242 no events in 237 unexpected events in 239 PMTs See photomultiplier tubes populations sorting 52 90 161 162 troubleshooting 235 242 power buttons 24 cytometer 24 100 fluidics cart 3 5 laser 24 precision modes 53 57 See also sort precision modes preferences user 143 pressure ar 5 sample 30 sheath 29 64 sort default values 85 troubleshooting 244 primary laser verifying area scaling 312 priming fluids 113 184 Index 333 printing sort reports 96 165 worksheets 144 pulse electronic 41 purging filters 190 Purity Masks 55 mode 57 162 Q quality control QC about 308 beads 310 experiment 97 particles 261 tracking results 324 R reagents parts list 262 recording data 138 142 during sorting 165 QC data 319 refilling ethanol shutdown tank 111 plastic containers 112 sheath tank 109 removal tool deflection plate 219 removing defle
111. clean 244 longpass filters 36 M maintenance AMO 283 scheduled 182 temperature control option 304 unscheduled 204 managing aerosols 16 Masks about 53 default precision modes 57 Phase 56 Purity 55 Yield 54 Master DAQ overflow error 245 micrometer dial 23 157 160 mirrors dichroic 36 mismatch dialog configuration 80 cytometer settings 134 modes defining 59 deleting 59 332 BD FACSAria II User s Guide sort precision 53 57 monitoring sorts 94 165 N ND filters 39 223 257 Near UV laser about 265 configurations 266 269 optical filters 268 side population guidelines 272 specifications 267 troubleshooting 274 neutral density filters 39 223 257 nozzles See also closed loop nozzle about 12 breakoff patterns 107 changing 205 cleaning 206 integrated 12 lever 103 205 O ring 208 258 sizes 85 205 spare 258 standard 13 208 O octagon about 21 22 34 default filters 252 opening aspirator drawer 50 92 164 sample injection chamber 10 Optical Filter control 50 51 optical filters about 35 changing 222 cleaning 223 custom 222 default setup 252 Near UV laser 268 optics about 18 collection 20 default setup 252 emission 252 excitation 18 20 251 fiber 19 filter holders 257 reordering 256 stream viewing 23 optimizing cytometer performance 308 cytometer settings 131 drop delay 156 PMT voltages 128 streams 170 ordering supplies 255 O ring nozzle 208 258 sample injection
112. ct Cytometer gt View Configurations which opens the Cytometer Configuration window in the CS amp T module See Figure 3 5 on page 70 NOTE When you select Cytometer gt View Configurations the cytometer disconnects from the BD FACSDiva interface and connects to the CS amp T interface While the CS amp T module is running BD FACSDiva is in a holding mode and does not accept any user input When CS amp T is closed BD FACSDiva becomes active again 2 Select a cytometer configuration that contains appropriate settings for the samples you are running A For accurate data results the cytometer optics must match the current cytometer configuration Also the sheath pressure and sort setup must match the settings in BD FACSDiva 3 Once the appropriate configuration is selected click OK in the Cytometer Configuration window to close it 4 Close the CS amp T application by selecting File gt Exit The BD FACSDiva interface is now active and accepting user input See Figure E 1 and Figure E 2 for error messages that can occur if the sort setup or sheath pressure are different between the cytometer configuration in CS amp T and the values in BD FACSDiva Figure E 1 Possible error message when closing CS amp T CST Mismatch The settings from CST are different from those on the cytometer Do you want to use the CST values o Don t show this message again for current login session Use CST Settings Keep Current Settings
113. ction plates 219 ferrules 198 200 filters 191 222 223 sample line filter 212 replacement parts 260 replacing closed loop nozzle tubing 211 tubes 166 334 BD FACSAria II User s Guide reports cytometer status 72 printing 96 165 sort 94 96 results troubleshooting 236 rotating sort block 14 206 running performance check 120 S safety general 25 sample agitation 65 core diameter 31 flow 28 30 injection chamber about 9 lubricating O ring 220 sample line 213 interrogation 11 line backflush 184 changing 194 filter 212 213 pressure 30 temperature 66 100 tubes replacing 166 samples heating 66 100 running 142 satellites drop 106 Save Conflicts 91 162 saving application settings 130 sort conflicts 91 162 scaling area adjusting 310 primary laser 312 scatter light 32 parameters distorted 241 second laser adjusting delay 317 sensitivity cytometer performance 249 settings See also application settings optimizing 131 setup beads 118 compensation 137 values sort 85 86 sheath flow 29 fluid 108 line 189 pressure 29 64 reservoir autoclaving 187 sheath filter changing 192 purging 191 sheath probe containment device 186 removing 185 sheath tank capacity 108 changing air filter 203 changing filter 192 connectors 109 refilling 109 required location 111 shutdown tank ethanol capacity 108 refilling 111 shutting down computer 177 181 daily procedure 176 fluidics 64 176 178 l
114. cudrop 633 nm red diode laser provided for quick drop delay Automatic drop determination in software 250 BD FACSAria II User s Guide Excitation Optics Laser Specifications The following class 3B lasers are mounted on the cytometer Table 8 5 Laser specifications Illumination Laser single line laser Wavelength Power Manufacturer Model nm mw Z Coherent Sapphire 488 20 488 13 E 5 JDS Uniphase 1144 P 633 11 2S Point Source iFlex 2000 P 1 405 405 30 0 65 10 StokerYale Lasiris SNF 701L 660 35 1 deg 660 35 a Measured out of fiber optic cable NOTE For specifications on the Near UV laser 375 nm see Specifications on page 267 Chapter 8 Technical Specifications 251 Emission Optics Table 8 6 Emission optics specifications Optical coupling Quartz cuvette flow cell is gel coupled by refractive index matching optical gel to the fluorescence objective lens for optimal collection efficiency Numerical aperture is equal to 1 2 filters Forward scatter detector and Photodiode detector with a 488 10 bandpass filter Side scatter detector Photomultiplier with 488 10 bandpass Fluorescence detectors and filters Three fixed fiber apertures 200 micron separation Detector Arrays Table 8 7 Default setup for detector arrays Detector Array PMT LP Mirror BP Filter Inten
115. d 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 reflect 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 For the dichroic carefully assemble 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 cytometer configuration to identify which PMT identified by laser color and letter will detect the emitted light See Custom Configurations on page 74 for instructions BD FACSAria II User s Guide Cleaning the Optical Filters Optical filters should be inspected occasionally and cleaned as necessary The frequency will depend on how often the filters are handled AN 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 cotton swabs optical lens paper
116. d 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 i i J Laser beams Laser beams Low flow rate High flow rate 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 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 Chapter 2 Theory of Operation 31 Signal Generation 32 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 r
117. d replaced if necessary See the indicated sections for the following maintenance procedures Table 6 3 Unscheduled maintenance Procedure Recommended Frequency Changing the Integrated Nozzle on page 205 As needed for different sized particles Cleaning the Integrated Nozzle on page 206 When stream irregularities indicate that the nozzle is clogged Using the Standard Nozzle on page 208 Use this for changing or cleaning the standard nozzle as needed Temporary Replacement of a Seal on page 208 As needed when seal is lost or damaged in an integrated nozzle Installing an O ring in a Standard Nozzle on page 209 As needed when O ring is lost or damaged Closed Loop Nozzle Maintenance on page 210 As needed Installing or Removing a Sample Line Filter on page 212 When the sample line filter needs to be installed or changed Changing the Pinch Valve Tubing on page 214 As needed Cleaning the Camera Windows on page 217 When smudges appear in the Breakoff or Side Stream windows Removing the Deflection Plates on page 219 As needed to clean the deflection plates Lubricating the Sample Injection Chamber O Ring on page 220 As needed when the O ring is dry Using Custom Optical Filters on page 222 As needed Cleaning the Optical Filters on page 223 As needed when changing a filter Removing or Installing the FSC ND Filter on page 223 As needed
118. dard Nozzle on page 13 e Sort Block on page 13 e Sort Collection Chamber on page 17 Figure 1 7 Main fluidics components Pinch valve Cuvette Nozzle holder flow cell Sample injection Sort block rae door Deflection plates warning light Sort collection chamber Sample loading port 8 BD FACSAria II 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 63 You can view the amount of fluid remaining in your sample tube by pressing the chamber light button shown in Figure 1 8 A Do not use the chamber light for long periods with samples stained with light sensitive reagents Figure 1 8 Sample injection chamber Sample Ay Chamber light injection button chamber ha Splash shield Tube holder Le Emergency stop button Loading port Chapter 1 Cytometer Components 9 Tube Holders A variety of tube holders are provided with the cytometer to accommodate tubes from 15 mL centrifuge tubes to 1 0 mL microtubes Figure 1 9 For a list of compatible tubes see Labware on page 263 To load a tube install the appropriate size tube holder onto
119. ded Fluorochrome or Dye Laser Octagon A 735 780 60 PE Cy 7 488 nm blue laser 655 695 40 PerCP Cy5 5 or PI 675 20 PerCP PE Cy5 C 610 616 23 PE Texas Red D 556 585 42 PE or PI E 502 530 30 FITC GFP Alexa 488 F 488 108 Side scatter SSC Trigon A 735 780 60 APC Cy7 APC H7 633 nm red laser B 660 20 APC 252 BD FACSAria II User s Guide Detector Arrays Table 8 7 Default setup for detector arrays continued Detector Array PMT LP Mirror BP Filter Intended Fluorochrome or Dye Alexa Fluor 430 Am Cyan Laser Trigon A 502 530 30 405 nm violet laser B 450 40 Cascade Blue Pacific Blue DAPI Hoechst Alexa Fluor 405 a The optical holder for the 488 10 BP filter also includes a 1 0 ND filter Chapter 8 Technical Specifications 253 Fluidics Cart Specifications Table 8 8 Fluidics cart specifications Dimensions Height 66 cm 26 in Width 81cm 32 in Depth 66 cm 26 in Weight 81 7 kg 180 lb fluidics cart only excluding tanks Facilities No air supply or vacuum required Optional Can be connected to facility air supply Containers e One 10 L stainless steel sheath tank e One 5 L stainless steel ethanol shutdown tank e One 10 L plastic waste container e Three 5 L plastic fluid containers for bleach DI water and ethanol 254 BD FACSAria II User s Guide Appendix A Supplies and Consumables Th
120. device that evacuates the sort collection chamber during sorting The AMO is equipped with an ultra low penetrating air ULPA filter to trap aerosolized particles See Appendix C BD Aerosol Management Option on page 275 for complete information on using this option BD FACSAria II User s Guide Sort Collection Chamber Collection devices are installed in the sort collection chamber to collect sorted samples The collection devices are designed in two pieces with a universal top that can be used with different tube configurations Figure 1 15 shows the types of collection devices that are available Figure 1 15 Sort collection devices i i e oo oo OO CO Universal top Four way 1 mL Four way 12 x 75 mm optional f o o i c Oe pE F Two way 12 x 75 mm Four way 1 5 mL Eppendorf Two way 15 mL An automated cell deposition unit ACDU that sorts into multiwell plates and onto microscope slides is available as an option See Figure 1 16 on page 18 BD Biosciences also offers a temperature control option to maintain the temperature of sorted samples during sorting The sort collection chamber door should be kept closed when sorting into a plate The door keeps the chamber free of dust and other airborne particles and seals the chamber during aerosol evacuation for cytometers equipped with the AMO Chapter 1 Cytometer Components 17 Figure 1 16 Plate Loader on ACDU Optics System The
121. different from the sort setup setting The gap values and sensitivity algorithms are optimized for particular nozzles and are not suitable for other nozzle sizes The system performance could be unstable Table 3 2 Default sort setup values Setting 70 micron 85 micron 100 micron 130 micron Sheath Pressure 70 45 20 10 Amplitude 60 32 12 24 Frequency 87 47 30 12 Drop 1 150 150 150 150 Gap upper limit 6 14 7 17 10 21 12 21 Attenuation Off Off Off Off Drop Delay 47 00 30 00 27 00 16 00 Far left voltage 100 100 80 60 Left voltage 40 35 30 20 Right voltage 40 35 30 20 Far right voltage 100 100 80 60 Plate voltage 4 500 4 000 2 500 2 000 2nd Drop 20 20 10 0 3rd Drop 10 10 5 0 4th Drop 0 0 0 0 86 BD FACSAria II User s Guide Table 3 2 Default sort setup values continued Setting 70 micron 85 micron 100 micron 130 micron 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 The Sort Layout window contains 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
122. e Drop Delay Manual Method on page 154 for more information 58 BD FACSAria II User s Guide Defining New Precision Modes Default Precision modes cannot be edited or deleted However you can create new modes and then select them from the Precision Mode menu 1 Select Sort gt Sort Precision amp Sort Precision Precision Mode v Yield Mask Purity Mask Phase Mask Single Cell 2 Click Add and the current sort mode is duplicated and the Mask fields are enabled 3 Optional Change the name of the mode in the Precision Mode field 4 Enter values for the Yield Purity and Phase Masks 5 Select the Single Cell checkbox if needed 6 Click Close The new mode is added to the Precision Mode menu To delete a mode select it from the Precision Mode menu and click Delete Chapter 2 Theory of Operation 59 THIS PAGE INTENTIONALLY LEFT BLANK 3 Using BD FACSDiva Software Many BD FACSAria II cytometer functions are controlled using BD FACSDiva software This chapter provides a general overview of the workspace components and describes software controls that are unique to the BD FACSAria II cytometer For an in depth description of software components not described in this chapter see the BD FACSDiva Software Reference Manual The following topics are covered in this chapter e Workspace Components on page 62 e Cytometer Controls on page 63 e Sorting Controls on page 83 e Templates on page 97 6
123. e cece eee e eens 81 Sorting Controls erpiar e E A Pelee bak ae Lad haw aaa 83 Sort Menu ssc ce awa Swan Saeed ews oes awe a eee yee 84 SOFt Setup ectht saa as Mae yeas ania eae ae eA eis ask aa es 85 Sort Layout 64 434s BSAA RAISE ENE A ARERR OSES eS 87 Sort REPO ssid i we ate a Se da Sao ae alee ae EN 94 Templates ta mirra ews a4 baw eee wee eh E ee Geeta eed hee Saas 97 vi BD FACSAria II User s Guide Chapter 4 Running Samples Cytomet r Startup 4 s 200 He eV Pew pest Ove gees Performing Fluidics Startup 0 eee Starting the Stream 1 eee eee ee ees Setting Up the Breakoff 0 0c eee eee Setting Up the Fluidics Cart 000005 Checking Cytometer Performance 0 005 Preparing the CS amp T Workspace 0 Preparing the CS amp T Beads 0c ee eee Running a Performance Check Reviewing the Results 0 0 0 e eee ueee Application Settings 0 ee cece eee eee Creating Application Settings 4 Data Gollection s0s none gees debere ee dele ates Setting Up the Workspace 0 000 eee eee Calculating Compensation 0000 Data Recording and Analysis 0 0 00 eeeuee Setting Up the Experiment 0 005 Setting Up the Global Worksheet Recording Data 0 0 0 cece eee e eens Analyzing Data 2 cece ccc eee eens Performing a Batch Analysis 4 C
124. e threshold to remove most of the debris without cutting off the singlet population Figure 4 23 Adjust the P1 gate to surround only the singlets Figure 4 23 Right click the P1 gate and select Apply to All Compensation Controls The P1 gate on each stained control worksheet is updated with your changes Click Record Data When recording is finished click Unload and remove the unstained control tube from the cytometer Do not change the PMT voltages after the first compensation control has been recorded In order to calculate compensation all controls must be recorded with the same PMT voltage settings If you need to adjust the PMT voltage for a subsequent compensation control you will need to record all compensation controls again Install the next tube onto the cytometer and repeat steps 8 and 9 until data for all stained control tubes has been recorded Double click the first stained control tube to display the corresponding worksheet BD FACSAria II User s Guide 12 Verify that the Snap to Interval gate encompasses the positive population Figure 4 24 Adjust the gate if needed Figure 4 24 Gating the positive population FITC Stained Control FITC Stained Control s SSC A amp 1 000 0 190 160 200 250 Count 50 100 150 200 250 FSC A x 1 000 FITC A 13 Repeat steps 11 and 12 for the remaining compensation tubes 14 Select Experiment gt Compensation Setup gt Calculate Compensation
125. ea scaling factors should be verified for each experiment performed on the cytometer 1 In the Browser right click Global Sheet1 and select Apply Analysis Template In the Template dialog select the Area Scaling worksheet and click OK Create a new specimen by clicking the New Specimen button on the Browser toolbar Expand the new specimen then click to set the current tube pointer to Tube_001 Install the FITC positive control tube onto the loading port and click Load in the Acquisition Dashboard 124 BD FACSAria II User s Guide 6 Adjust the FSC and SSC voltages to place the particles on scale See Figure 4 14 on page 125 Figure 4 14 Adjusting FSC and SSC voltages Cytometer FACSAria 1 Parameter aaron 7 Adjust the P1 gate around the population of interest in the FSC vs SSC plot See Figure 4 15 Figure 4 15 FSC and SSC plot 200 250 1 000 8 Adjust the FSC area scaling a Click the Laser tab in the Cytometer window See Figure 4 16 Chapter 4 Running Samples 125 Figure 4 16 Adjusting FSC area scaling Cytometer FACSAria 1 b Adjust the FSC area scaling factor until the FSC A signal matches the FSC H signal See Figure 4 17 e Increase the area scaling factor if the FSC A signal is lower than FSC H e Decrease the area scaling factor if the FSC A signal is higher than FSC H c View the result of your change in the hi
126. eate a custom configuration is to copy and edit a base configuration You can also create a new blank configuration 1 Log in to the software as an administrator 2 Select Sort gt Sort Setup and select the setup mode that matches the nozzle size you are using 3 Verify that the correct nozzle is installed then start the stream 4 Select Cytometer gt CST The Cytometer Configuration window appears See Figure 3 5 on page 70 For users with administrator access the window displays the Parameters and Filters and Mirrors tabs which are not visible to users with operator access NOTE When you create a new configuration in CS amp T you need to run a new baseline for that configuration Once the baseline is established you cannot edit any of the settings in the configuration BD FACSAria II User s Guide Adding Parameters Filters and Mirrors Before creating a custom configuration verify that the necessary parameters filters and mirrors required for the custom configuration are defined 1 To add new parameters do the following in the Parameters tab a Click Add b Enter a parameter name in the field provided PE PE Cy7 PerCP PerCP Cy5 5 PE Texas Red PI Qdot Qdot 525 Qdot 565 Qdot 585 Qdot 605 Qdot 655 Uv u 2 iolet1 iolet2 Enter new parameter 2 To add filters or mirrors do the following in the Filters and Mirrors tab a To add a filter click Add under the Filter list To
127. ed it could affect the flow rate calibration 110 BD FACSAria II User s Guide 8 Figure 4 6 Sheath tank location label ATTENTION SHEATH TANK W LOCATED HERE during instrument operation to maintain calibration Connect the air line The system is ready to run again Refilling the Ethanol Shutdown Tank The fluid level in the stainless steel ethanol shutdown tank should be checked before starting the fluidics shutdown procedure and refilled when low This tank does not have a sensor so it must be checked manually Figure 4 7 Ethanol shutdown tank connectors Fluid connector Ethanol filter Pressure relief valve Air connector Cover knob Turn off the stream If it is connected disconnect the air line on the ethanol shutdown tank Pull up on the ring of the pressure relief valve to release pressure from the tank Make sure the tank is fully vented Chapter 4 Running Samples 111 4 Unscrew the tank cover knob and remove the cover 5 Fill the tank with ethanol to the level shown in Figure 4 8 6 Replace the cover and tighten the knob Make sure the large O ring on the inside of the cover is seated correctly and has not slipped out of position The tank can leak if the cover is not secured properly 7 If you are going to perform fluidics shutdown connect the air line Figure 4 8 Ethanol shutdown tank fill level Fill level Re
128. ed Nozzle on page 205 for instructions Sorting paused because actual Drop 1 value is out of range Wait until the Sweet Spot adjusts the amplitude to achieve the Drop 1 target If this happens repeatedly during sorting there might be debris in the nozzle or flow cell See Troubleshooting the Stream on page 226 for suggestions 234 BD FACSAria II User s Guide Sorting Troubleshooting continued Observation Fanning around center or side streams Possible Causes Nozzle inserted improperly Recommended Solutions Turn off the stream Remove the nozzle and ensure that the seal or O ring is in place Re insert the nozzle and slide the nozzle in until it stops then close the locking lever Sweet Spot is 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 57 2nd 3rd or 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 Integrated Nozzle on page 205 Sort button disabled Current tube pointer not set to current tube Click to move the current tube pointer to the appropriate tube Population not listed in Add menu on sort layout Population defined using snap to gate Redefine the population usi
129. efault Cytometer Name Vali SerialNumber P222300090 igurations Parameters Filters and Mirrors eS 3 laser 9 color 5 2 2 Configuration 5 BD default B Base Configurations f L E 3 laser 9 color 5 2 2 Configuration 5 01 02 07 Blue Laser 488nm FSC Red Laser 633nm Violet Laser 407nm Alera Fltor 30 PE Texas Red Hoechst Cacak Bhe IL s All Blue Red Violet Sheath Pressure psi 70 00 Window Extension ys 2 00 Nozzle Size u 70 Set Configuration Print Export Before you start any experiment verify that the cytometer configuration contains appropriate parameters for the samples you are running and that the cytometer optics match the current configuration If needed you can add or modify parameters in an existing configuration or define a custom configuration as described in Custom Configurations on page 74 A For accurate data results the cytometer optics must match the current cytometer configuration 70 BD FACSAria II User s Guide Selections in the Cytometer Configuration window determine which parameters are available for your experiment When more than one parameter is available for a detector the first parameter is listed by default To select a different parameter click the name in the Parameters tab of the Cytometer window in BD FACSDiva software and select a different parameter from the menu Cytometer FACSAria 1 Parameter Volta
130. eful with tandem conjugates due to lot to lot variation See the BD FACSDiva Software Reference Manual for more information about this feature 134 BD FACSAria II User s Guide A compensation specimen is added to the experiment along with a stained control tube for each compensation control Expand the specimen to view all tubes Worksheets containing appropriate plots are added for each compensation tube B q Compensation Controls 8S Cytometer Settings J Unstained Control J FITC Stained Control Jf PE Stained Control J PercP cy5 5 Stained Co J APC Stained Control Oe o oo Calculating Compensation The unstained control will be used to verify the settings for FSC SSC and FSC threshold and to gate the population of interest 1 2 Install the unstained control tube onto the cytometer Expand the compensation specimen in the Browser Set the current tube pointer to the unstained control tube and click Load Verify that the population of interest is displayed appropriately on the FSC vs SSC plot Adjust if needed See Figure 4 23 Since the application settings have already been optimized for your sample the cytometer settings should require little or no adjustment Figure 4 23 Voltages adjusted Unstained Control s 8 3 3 amp lt a ao 150 200 250 FSC A x 1 000 Chapter 4 Running Samples 135 136 10 11 Click the Threshold tab and adjust the FSC threshold if needed Set th
131. elative differences in the internal complexity or granularity of the cells or particles Side scatter Forward scatter Light source BD FACSAria II User s Guide Fluorescent Signals 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 known 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 8 7 on page 252 for the corresponding detectors Figure 2 5 Emission spectra of commonly used fluorochromes 100 Cascade Blue FITC Alexa Fluor 430 Pacific Blue RPE PI APC PerCP PerCP Cy5 5 PE Cy7 Normalized Intensity W 400 500 600 700 800 Wavelength nm Chapter 2 Theory of Operation 33 Signal Detection 34 From the cuvette flow cell sca
132. en the sort collection chamber door if needed The sort block door must be closed to install the tube holder b Slide the holder into the slotted fittings below the sort aspirator drawer then close the sort collection chamber door AA Before installing the collection tube holder ensure that an O ring is 152 installed in the groove between the two sections of the tube holder The O ring minimizes the chance of aerosols escaping It can be found in the accessory kit Catalog No 337897 3 Turn on the deflection plates Click the Voltage button in the Side Stream window Figure 5 1 on page 153 The voltage warning light illuminates indicating that the plates are charged BD FACSAria II User s Guide A amp A 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 open Figure 5 1 Turning on the deflection plates Voltage Test Sort Optical Filter _ Attenuation Waste Drawer Drop Delay 39 57 4 fy ja m w Voltage button 91 E LJ Voltage Center Plate voltage 5 000 f 2nd Drop 20 3rd Drop 10 2 4thDrop 0 Phase Make sure the center stream image does not move after the plates are turned on Major movement of the center stream could indicate that the plates or area around
133. end the life of your ULPA filter 1 Open the sort collection chamber door and remove the air filter 288 BD FACSAria II User s Guide Push the filter toward the left side of the door until you can grasp it and then slide the filter out of the door Figure C 11 Removing the air filter Install a new air filter in the door Slide the new filter in from the left NOTE Make sure to install the filter with the grid side facing up when the door is open Grid side faces up when door open Non grid side faces out when door closed Appendix C BD Aerosol Management Option 289 Troubleshooting The tips in this section are provided to help you troubleshoot issues that arise when using the BD Aerosol Management Option For cytometer specific troubleshooting see Troubleshooting on page 225 If additional assistance is required contact your local BD Biosciences technical support representative or supplier A If any of the following are observed assume the AMO is not evacuating properly and do not open the doors to the sort chamber Control Panel Troubleshooting Observation Evacuator indicator lights off Possible Cause Evacuator power cord unplugged Recommended Solution Connect the evacuator power cord to the power source Evacuator power switched off Switch on the evacuator main power See Figure C 4 on page 280 Evacuator motor failure Contact your local BD technical support repres
134. ength 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 the Secondary Sample Line This section describes changing the secondary sample line see Figure 6 12 on page 194 There is a different fitting at each end of the tubing so the procedure is divided into two sections Pinch Valve End The procedure to replace the secondary sample line at the pinch valve end is the same as for the primary sample line except the replacement line is a 7 inch length See Assembling the Collet Fitting at the Pinch Valve on page 195 Chapter 6 Shutdown and Maintenance 199 Flow Cell End 1 Turn the stream off if needed 2 Unscrew the connecting nut at the top of the flow cell and slowly pull out the sample line See Figure 6 17 Figure 6 17 Secondary sample line insertion ___ Secondary sample line Flow cell 3 Ensure that a cone shaped ferrule is attached to the sample line If the ferrule was left behind in the flow cell fitting gently push 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 might need to replace the ferrule If the ferrule is damaged replace it with a spare included in the accessory kit 4 Slide the ferrule and nut off the end o
135. entative Circuit breaker tripped Depress the circuit breaker on the rear of the evacuator into its original position Site power failure Turn off the evacuator power switch and wait for site power to be restored Evacuator indicator lights pulsing Erratic power source Plug the power cord into a different outlet 290 BD FACSAria II User s Guide Control Panel Troubleshooting continued Observation Possible Cause Recommended Solution Arrow keys not Improper operation Push each button firmly before responding removing your finger from the control Defective membrane panel Contact your local BD technical support representative Red filter life Approaching 180 hours of Monitor the light When it indicator light on filter use blinks change the filter Filter life not reset after After changing the filter press filter change and hold the Filter Life Reset button until the 100 indicator light is lit Red filter life Filter used over 180 hours Replace the filter indicator blinking Appendix C BD Aerosol Management Option 291 Filter Flow Gauge Troubleshooting Observation Zero reading on filter flow gauge Possible Cause Power off Recommended Solution Press the power button on the membrane panel of the evacuator Filter defective Replace the filter Filter improperly seated in evacuator Reseat the filter with the evacuator power off
136. epending on the state of the system and the sort setup selected 1 Start the stream a Click the Sorting button on the Workspace toolbar to display the Breakoff and Side Stream windows b Click the Stream button in the Breakoff window to turn on the stream 2 Open the sort block door and check the stream The stream should flow smoothly from the nozzle into the center of the waste aspirator If the stream is flowing but is unsteady check for bubbles in the flow cell If you see bubbles turn off the stream wait for 10 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 see Troubleshooting the Stream on page 226 3 Close the sort block door NOTE If you encounter bubbles in the sheath fluid stream at the 130 micron sort setting perform a sample line backflush for approximately 1 minute Select Cytometer gt Cleaning Modes gt Sample Line Backflush To prevent this the sheath tank should be depressurized while not in use to prevent air from dissolving into the sheath solution 104 BD FACSAria II User s Guide Setting Up the Breakoff Establishing a stable drop pattern in the breakoff window is an important step in getting optimal results from the system See Breakoff Window on page 46 for more information on the parameters and controls in the breakoff window 1 Adjust the Ampl slider until the drop breakoff is approximately in the top
137. er Figure 1 12 Chapter 1 Cytometer Components 13 Figure 1 12 Sort block with door open Deflection plates Adjustment screws Aspirator Aspirator drawer Sort collection device 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 different nozzles 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 both sides of the deflection plates and rotating the sort block An Allen wrench is provided in the accessory kit Tighten the screws when the stream is re centered in the aspirator 14 BD FACSAria II 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 49 A red warning light is illuminated whenever the plate voltage is on Figure 1 14 on page 16 A amp Aa 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 doo
138. er button on the Workspace toolbar Check the fluid levels at the bottom of the window Figure 4 2 Figure 4 2 Fluid levels in the Cytometer window Sheath Waste DI Bleach Ethanol Note Roll the mouse pointer over Cytometer Connected fluid indicators to show fluid type To service the fluid containers see Setting Up the Fluidics Cart on page 108 and Emptying the Waste Container on page 114 Performing Fluidics Startup This section describes the fluidics startup procedure The status of the fluidics system is displayed in the bottom right corner of the main window Chapter 4 Running Samples 101 1 From the BD FACSDiva Cytometer menu select Fluidics Startup The following window appears x Gp Ensure the alr and Nuki ines are isconnected from ETOH pong tank and connected to the sheath tank 2 Verify that the air and fluid lines are disconnected from the ethanol tank and connected to the sheath tank then click Done See Figure 4 3 A Caution Do not run fluidics startup with the air and fluid lines connected to the ethanol shutdown tank This can cause damage to the system Always verify that the lines are connected to the sheath tank Figure 4 3 Air and fluid lines connected to the sheath tank Top view Side view Fluid line Sheath filter Ethanol l filter Air line Ethanol shutdown Sheath tank tank 3 Verify that a closed loop nozzle is installed in the flow
139. er window b Select the voltage field for the parameter you need to adjust c To adjust the values you can e Click the up and down arrows to adjust the voltages in increments of 1 e Drag the sliders to adjust the voltages e Press the Ctrl key while clicking the on screen arrows to increase or decrease in increments of 10 e Press the Ctrl key while pressing the keyboard arrows to increase or decrease in increments of 10 Appendix E OC Using BD FACSDiva Software 313 50 100 1 50 200 FSC H 250 1 000 7 Adjust the FSC area scaling until the FSC A signal matches the FSC H signal if needed a Click the Laser tab in the Cytometer window and check the FSC area scaling value Cytometer FACSAria 1 Cytometer Connected e If the FSC A signal is lower than FSC H increase FSC area scaling e If the FSC A signal is higher than FSC H decrease FSC area scaling 314 BD FACSAria II User s Guide Figure E 4 FSC area scaling factor set incorrectly left and correctly right Rainbow beads High Sort Setup Rainbow beads High Sort Setup J i g E zS 5 5 3 f og 5 A amp J TIT TT TTT 50 100 150 200 250 50 100 150 200 250 FSC H 1 000 FSC H amp 1 000 Rainbow beads High Sort Setup Rainbow beads High Sort Setup o1 z E er 5 3 5 a J 3 Od A Torr m mr mr 0 100 150 200 250 50 100 150 200 250 FSC A amp 1 000
140. es BD FACSRinse detergent BD Biosciences 340346 BD FACSClean solution BD Biosciences 340345 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 See the BD Biosciences Immunocytometry Products Catalog or the BD Biosciences website bdbiosciences com b US Patent Nos 4 654 312 4 902 613 5 098 849 262 BD FACSAria II User s Guide Labware Item Supplier Catalog No 1 mL microtubes Bio Rad Laboratories 223 9391 800 424 6723 1 000 per box 1 5 mL Eppendorf tubes Various 5 mL polystyrene test tubes BD Biosciences 12 x 75 mm BD Falcon 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 e Polypropylene 50 bag e 352196 e Polypropylene 125 bag e 352096 e Polypropylene 50 rack e 352097 e Polystyrene 125 bag e 352095 e Polystyrene 50 rack e 352099 Appendix A Supplies and Consumables 263 THIS PAGE INTENTIONALLY LEFT BLANK Appendix B Near UV Laser Option The Near UV laser option for the BD FACSAria II system fe
141. es 113 Emptying the Waste Container Empty the waste container daily and when the fluid indicator shows the waste is getting full To prolong the life of the container we recommend that you switch to the alternate container each time the waste is emptied AA AA 1 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 of waste in accordance with local regulations Use proper precautions and wear suitable protective clothing eyewear and gloves Change the waste container cap every month to prevent container pressurization To order new replacement caps BD Part No 33885407 pack of 12 contact your local BD Biosciences representative Figure 4 10 Waste container details Waste ports behind handle Disposable cap Trap Waste container Disconnect the waste container s sensor and fluid line connectors from their respective ports on the fluidics cart 114 BD FACSAria II User s Guide Sensor Waste fluid lines A The waste container can become pressurized when the cytometer is running Always disconnect the container from the fluidics cart before you empty it Wait at least 1 minute for pressure to dissipate before you open the container 2 Remove the disposable waste cap large sized cap and attac
142. ettings used on the cytometer are restored except the Stream and Sweet Spot controls which always default to off For more information see Sort Setup on page 85 Table 2 1 Breakoff window controls Control Description Stream button Turns the stream on and off EA 70 micron x z Stream x By Sweet Spot off Sweet Spot Enables automatic adjustment of the drop drive button amplitude to maintain the stability of the breakoff point A Ra When the Sweet Spot is on the Amplitude and On of 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 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 Determines the number of drops formed per second and 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 Chapter 2 Theory
143. f the APC A signal is lower than APC H e Decrease the area scaling factor if the APC A signal is higher than APC H Repeat step 1 through step 3 to adjust area scaling and laser delay for the violet or Near UV laser using the appropriate parameter instead of APC Recording QC Data The QC experiment template includes Browser elements as well as plots and instrument settings The first time you use the QC experiment template adjust the settings by placing the signals for each parameter to channel 100 000 The instrument settings you establish will be used for subsequent QC runs Appendix E OC Using BD FACSDiva Software 319 1 Display the QC worksheet by clicking the QC tab in the Worksheet window 2 Adjust the PMT voltage for each parameter to set the signal to approximately 100 x 103 Rainbow beads 032806 50 100 150 200 250 FITC A amp 1 000 3 Set the flow rate to 1 0 in the Dashboard 4 Ensure that the current tube pointer is still set to the first tube and then click Record Data Once the tube has finished recording the tube icon changes to a tube witha disk Also the tube will now have cytometer settings associated with it Lh March 2007 QC Cytometer Settings Global Worksheets Ej S Rainbow beads B 01maro7 Tube with data 8S Cytometer Settings J c2maro7 _____ Tube without data 5 After data has been recorded click Unload in the Dashboard and remove the tube 6 Optional
144. f the sample line 5 Slide the nut and then the ferrule onto the end of the new sample tubing Leave approximately 0 1 inch 0 25 cm of tubing extending out of the ferrule This length is referred to as the pilot as shown in Figure 6 16 on page 198 200 BD FACSAria II User s Guide 6 Insert the pilot tubing into its fitting at the top of the flow cell ensuring that the tubing reaches the intended pilot depth A Within 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 Finger tighten the nut at the top of the flow cell to secure the sample line A Do not overtighten the nut and do not use tools Over tightening the nut can kink or damage the tubing 8 Check the fitting connections at both ends to make sure they are not leaking Turn on the stream load a tube of DI water and make sure none of the fittings are leaking If needed unload the tube turn off the stream and tighten the fittings After tightening if leaking still occurs replace the ferrule A A Make sure all fittings are securely tightened If any fitting is loose the EX i tubing could detach during high pressure operation exposing the operator to potentially biohazardous sample spray Chapter 6 Shutdown and Maintenance 201 Changing the Air Filters The BD FACSAria II cytometer has two air filters one in the sort col
145. factor For proper cytometer operation change the sheath pressure by selecting an option from the Sort gt Sort Setup menu Do not adjust the pressure using controls in the Sheath Pressure dialog Note that the Sheath Pressure command is disabled when the Sweet Spot is on Sample Agitation Select Cytometer gt Sample Agitation to specify the speed at which samples are agitated You can select from one of the specified values or select Off to turn off agitation fev Sort Help Fluidics Startup Fluidics Shutdown Change Sample Filter Cleaning Modes gt Sheath Pressure Sample Temperature gt 300rpm Cytometer Details 200rpm View Configurations CST Performance Tracking LJ Catalogs Standby Chapter 3 Using BD FACSDiva Software 65 66 Sample Temperature Use the Sample Temperature command to set the temperature inside the sample injection chamber You can select from one of the specified values or select Off to turn off temperature control Figure 3 2 Figure 3 2 Setting the sample temperature Fluidics Startup hb Fluidics Shutdown Change Sample Filter Cleaning Modes gt Sheath Pressure Sample Agitation soff Cooling heating Cytometer Details 4C unit for sample 20c injection chamber 37C 42c view Configurations CST Performance Tracking LJ Catalogs U Standby The cooling heating unit is desi
146. fault state is in For more information see Aspirator Drawer on page 15 BD FACSAria II User s Guide Using Counters Counters provide ongoing status during sorting Counter fields cannot be edited To display fewer counters in the Sort Layout window click the View Counters button and select a menu option The corresponding counter is hidden Only counters with a checkmark next to the name are displayed Tube_001 Sort Layout_001 Device Precision Target Events Save Sort Reports Save Conflicts 2 Tube v Purity w Ask User v Oo Left 7 Right Sort Rate Confl Cnt Confl Rate Efficiency View Counters v Sort Rate v Conflicts Count v Conflicts Rate v Efficiency Counters display the following information e Sort Rate Number of events per second that met the sort criteria and were sorted e Conflict Count Number of events that met the sort criteria but were not sorted because of conflicts e Conflict Rate Number of conflicts per second e Efficiency Number of sorted events sort conflicts sorted events x 100 Chapter 3 Using BD FACSDiva Software 93 94 Monitoring a Sort During sorting each sort location field displays the number of actual sorted events Figure 3 12 When a target number is specified the field displays the actual number of events along with the number of target events A progress bar appears behind the sort rate counter field sh
147. fferent from optical setup Verify that the cytometer optics match the current cytometer configuration No sample in the tube Add sample to the tube or install a new sample tube Chapter 7 Troubleshooting 237 Acquisition Troubleshooting continued Observation No events in plots after clicking Acquire Data continued Possible Causes Sample is not mixed properly Recommended Solutions Increase the sample agitation rate See Sample Agitation on page 65 Sample line is clogged Perform a sample line backflush See Sample Line Backflush on page 184 If necessary change the sample line Sample filter is clogged Replace sample filter Threshold not set to the correct parameter usually FSC Set the threshold to the correct parameter for your application Multiple threshold parameters not set correctly 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 Calculating Compensation on page 135 Optical filter s not completely seated Make sure the filters are pushed all the way in FSC area scaling is incorrect Ensure that the FSC H matches the FSC A value No fluorescence signal Current cytometer configuration different from optical setup Verify that the cytometer optics match the current cytometer configuration Wrong filter installed
148. figuration 8 BD default Blue Laser Red Laser 488nm 633nm FSC APC CVT APC Violet 375 UY Laser Alea Fior 30 PE Texas Red All Blue Red Violet 375 uv Sheath Pressure psi 70 00 Window Extension us 2 00 Nozzle Size p 70 Appendix B Near UV Laser Option 269 Switching From the Violet to the Near UV Laser The system should be turned on and all cytometer startup procedures performed before using the Near UV laser See Cytometer Startup on page 100 for information on these procedures This section provides an overview of the steps involved in switching to the Near UV laser in a four laser system 1 Place the Near UV OFF Violet laser switch in the Near UV position This turns on the Near UV laser and turns off the violet laser Allow the Near UV laser to warm up for 30 minutes before recording data 2 Set the cytometer configuration to a custom configuration that includes parameters for the Near UV laser It is a good practice to include Near UV in the configuration name for example 70 70 Near UV This is especially helpful in a four laser system A Verify that the custom configuration contains the correct parameters for use with the Near UV laser The system will not perform correctly if this is not done 3 Ensure that the appropriate optical filters for Near UV operation are installed into the far left trigon See Figure B 3 on page 268 4 Make sure the cytometer ha
149. filling the Plastic Containers You can refill the plastic containers directly on the fluidics cart without detaching any lines or you can remove the container for refilling Note that during operation you can add fluid to a container 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 See Figure 4 9 If you do not need to move the container skip to step 2 112 BD FACSAria II User s Guide Figure 4 9 Plastic fluidics container Sensor Quick release connector i Large sized cap Remove the large sized cap from the container See Figure 4 9 Fill the container with the fluid indicated on the container label Replace the container cap and hand tighten it until it is fully closed If you disconnected the sensor and quick release connectors in step 1 a Reconnect the sensor and quick release connectors to their respective ports To ensure that the appropriate solutions are dispensed do not switch the tank positions Make sure the label on each container matches the labeled port on the fluidics cart b Prime the fluidics system From the BD FACSDiva menu bar select Cytometer gt Cleaning Modes gt Prime after Tank Refill In the dialog that appears select the fluid to prime then click OK Chapter 4 Running Sampl
150. 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 38 Chapter 2 Theory of Operation 37 Figure 2 10 Bandpass BP vs discriminating DF filters 100 BP 500 50 filter DF 500 50 filter ag Ss 50 2 g x 0 450 500 550 Wavelength nm 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 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 8 7 on page 252 38 BD FACSAria II User s Guide Neutral Density Filters Neutral density ND filters transmit a fixed percentage of light reducing the transmitted intensity of all wavelengths equally ND filters are neutral with respect to wavelength gt 10 100 ND1 The ND1 filter on the BD FACSAria II allows approximately 10 of the light to be transmitted You can find the ND filter in front of the FSC detector For applications involving small particles eg bacteria or platelets you might need to remove the FSC N
151. ft or right as a whole In general the slider rarely needs adjusting BD FACSAria II User s Guide Table 2 2 Side Stream window controls continued Control Description Plate Voltage field Adjusts the total voltage difference between the plates which determines the angle of stream deflection The voltage values change with different nozzle sizes 2nd 3rd 4th Drop Apply a correction factor for the drop charge as a percentage of fields the previous drop from 100 to 100 Phase field Adjusts the phase between drop generation and charging of the droplets from 0 360 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 Drop Delay Overview The BD FACSAria II cytometer 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 BD FACS 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 green F 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
152. g 245 THIS PAGE INTENTIONALLY LEFT BLANK 8 Technical Specifications e Cytometer Specifications on page 248 e Fluidics Cart Specifications on page 254 247 Cytometer Specifications Table 8 1 Cytometer specifications Cytometer dimensions Height 71 cm 28 in Width 122 cm 48 in Depth 71 cm 28 in Table optional Height 85 1 cm 33 5 in Width 266 7 cm 105 in Depth 134 6 cm 53 in Weight Approximately 181 4 kg 400 Ib Power requirements 100 115 230 VAC and 50 or 60 Hz Power consumption maximum 1 500 watts 248 BD FACSAria II User s Guide Environment Table 8 2 Required operating conditions Storage temperature 1 40 C 34 104 F Operating temperature 15 30 C 59 86 F Operating relative humidity 5 80 noncondensing Noise level Less than or equal to 62 dBA Facilities Performance Table 8 3 Performance specifications No special room requirements Fluorescence resolution Coefficient of variation PI Area of lt 3 0 full Go G peak for propidium iodide PI stained chicken erythrocyte nuclei CEN Coefficient of variation Hoechst Area of lt 3 5 full Go G peak for Hoechst stained CEN Fluorescence linearity Forward and side scatter sensitivity Doublet singlets ratio for CEN stained with PI 1 95 2 05 detected off the 488 nm laser or Hoechst 1 95 2 05 detected off the 405 nm laser or 3
153. ge SIN 8 O0 Cytometer Connected Chapter 3 Using BD FACSDiva Software 71 72 Cytometer Status Report The Cytometer Status Report provides a list of all cytometer settings at the time the report was created You must be connected to the cytometer to create the report In an open experiment click to set the current tube pointer and select Cytometer gt Cytometer Status Report The report is displayed in a separate window with a menu bar above the report header The header lists the cytometer name type serial number and the date and time the report was generated See Figure 3 6 on page 73 for a sample report For a full description of the Cytometer Status Report see the BD FACSDiva Software Reference Manual A BD FACSAria II cytometer report includes the following additional information user access privileges cytometer information cytometer settings and sorting settings The report can be printed or exported e The User Access Privileges section lists access settings for the current user e The Cytometer Info section lists values for laser delay area scaling window extension FSC area scaling sheath pressure and sample flow rate e The Parameters section displays settings for the current acquisition tube 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
154. gned to maintain the temperature of a sample tube It is not designed to cool or heat the sample 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 Click Stop Acquiring to keep the chamber closed and stop running the sample To maintain the temperature do not leave the chamber open for extended periods while changing sample tubes The sample temperature retains the last setting after startup For example if it was set to 20 C the last time the system was used then it will return to that setting the next time the system is started up BD FACSAria II User s Guide Fluidics Level Indicators BD FACSDiva software provides fluidics level indicators in the Cytometer window 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 level is below 20 of the tank capacity When this occurs the corresponding level indicator changes to black NOTE The stainless steel ethanol shutdown tank does not have a level sensor It must be checked manually Figure 3 3 Fluid level indicators Sheath Waste DI Bleach Ethanol Cytometer Connected When the sheath is low or the waste is full while the stream is running the corresponding indicator turns red and the following wa
155. h in the orange collar as you pull the tubing out of the port Clean the temperature control tube holders by wiping them down with an appropriate cleaning fluid for example 70 ethanol 5 bleach or deionized water Dry them with a lint free cloth before storage Recirculating the Water Tubing Inspect the tubing periodically for leaks plugs or contaminants If needed remove the tubing and clean it with an appropriate cleaning solution or replace the tubing Contact your BD Biosciences service representative for replacement tubing BD FACSAria II User s Guide Specifications Specifications for the recirculating water bath are as follows NOTE The following specifications are for the US version only The temperature control option includes the Lauda Ecoline cooling heating bath model RE 106 e Operating temperature range 20 to 120 C e Ambient temperature range 5 to 40 C e Heater power for 115 V 60 Hz 1 3 kW e Maximum flow rate at pump output of 5 17 L min e Maximum bath volume 4 6 L e Power consumption for 115 V 60 Hz 1 4 kW For more information see the manufacturer s documentation Appendix D Temperature Control Option 305 THIS PAGE INTENTIONALLY LEFT BLANK Appendix E QC Using BD FACSDiva Software This appendix contains information on performing the quality control procedure using BD FACSDiva software 307 Cytometer Quality Control Using BD FACSDiva Software Perform cytome
156. hapter 5 Sorting Setting Up for Sorting 0 0 cee eee eee Setting Up for Bulk Sorting 005 Determining the Drop Delay Manual Method Setting Up the Experiment 00005 Using Manual Drop Delay Contents vii Determining the Drop Delay Automatic Method 2 2 000 159 Overview of Auto Drop Delay 0 ccc ccc cee eee eens 159 Using Auto Drop Delay 0 cece nee ences 159 Sorting nates Sacha Saves a ES va aoe Das hae bebo bates Hie 161 Setting Up the Experiment 0 ccc ec cence eee ee nees 162 Starting and Monitoring the Sort 0 0c cece cee eee eens 164 Stopping and Resuming a Sort 0 cee cece eee eee eens 166 Pausing and Resuming a Sort 0 cece eee eee ene eens 167 Setting Up for Sorting Into a Plate or Slide 1 0 0 cece ee ee 168 Installing the Sorting Hardware 0 ccc ccc eee es 168 Setting Up the Stream d cc8 cf cca ica e i ee aiee e e ads cee ale4 170 Creating a Custom Device 1 cc ccc ee eee teens 172 Chapter 6 Shutdown and Maintenance 175 Daily Shutdown ceire eei bas bie o De acd eddie pe ee ee 176 Cleaning the Flow Cell 00 ccc ce teen n eens 176 Fluidics Shutdown ssc an Sia tae a eee ele ee ae ae eae a 178 External Cleaning esasa nana foo 0s ak te ace os averse eed ae aa ak 181 Scheduled Maintenance 0 0 cece cee eee nen e nen nees 182 Internal Cleaning oi ec a a ae hd Sc See aa
157. he cytometer and do not need adjustment A 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 See the following sections for more information about the flow cytometer e Fluidics Components on this page e Optics System on page 18 e Cytometer Electronics on page 24 e Emergency Stop Button on page 25 Fluidics Components When the fluidics system is activated the sheath fluid from the pressurized sheath tank is forced from the fluidics cart up 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 collection device within the sort collection chamber Chapter 1 Cytometer Components 7 The following fluidics components are described in this section See Figure 1 7 on page 8 For more information about fluidics see Fluid Movement on page 28 e Sample Injection Chamber on page 9 e Tube Holders on page 10 e Cuvette Flow Cell on page 11 e Integrated Nozzle on page 12 e Stan
158. he detector e To add more than one parameter label to a detector Crtl click the parameters in the Parameters list then drag them to the detector 78 BD FACSAria II User s Guide e To change a filter select and drag a new filter from the Filters list to the filter slot e To change a mirror select and drag a new mirror from the Mirrors list to the mirror slot Blue Laser 488nm FSC rme Filter slot FP PerCP Cy5 5 Mirror slot Verifying that the Configuration Matches the Sort Setup To verify that the sheath pressure in the sort setup matches the sheath pressure in the configuration 1 2 Select File gt Exit to exit CSXT Select Sort gt Sort Setup and select the sort setup that matches the nozzle size listed in your configuration Help 70 micron Sort Precision 85 micron a 100 ieron Select the sort setup that ed asta matches the nozzle size listed in your configuration Sheath Pressure psi 70 00 Nozzle Size p 70 v Select Cytometer gt Sheath Pressure and verify that the pressure matches the pressure in your configuration Enter a new sheath pressure if needed Chapter 3 Using BD FACSDiva Software 79 The new sheath pressure is automatically saved with the current sort setup mode Sheath Pressure Enter the same sheath pressure that was Sheath Pressure Adjustment a entered in the configuration Sheath Pressure Sheath Pressure psi 70
159. he number of tubes wells or spots in the collection device Chapter 3 Using BD FACSDiva Software 89 90 Select the sort precision mode from the Precision menu For more information see Sort Precision Modes on page 57 Enter the number of events to be sorted in the Target Events field Once entered the number of events can be selected from the Target Events menu For continuous sorting select Continuous from the Target Events menu Select one of the following options from the Save Sort Reports menu e Save None Sort reports are not saved e Save All Automatically saves a sort report each time the sort is stopped e Ask User Prompts the user each time the sort is stopped to select whether or not to save the sort report This is the default option This setting is saved with the sort layout Select the field s corresponding to the tube s well s or spot s where the population will be sorted and select a defined population from the Add menu When you click in a sort location field a menu appears allowing you to add delete or clear all populations in the field Figure 3 11 Figure 3 11 Adding populations to be sorted Global Sheet1 Sort Layout_001 X Device Precision Target Events Save Sort Reports Save Conflicts 2 Tube v Purity v Continuous v Ask User vi o Left Right P1 Continuous C P2 Sort Rate Delete Wrs Confl Cnt Clear All Hrs NA Confl Rate View Counters
160. hed trap from the container Place the assembly on the bench label side up AA Do not wet the cap on top of the trap If you see liquid inside the trap remove the drain plug and fully drain the liquid before you replace the plug Figure 4 11 on page 115 Figure 4 11 Draining liquid from the trap 3 Empty the waste container according to your standard laboratory procedures for biohazardous waste 4 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 bleach Chapter 4 Running Samples 115 5 Replace the waste trap and attached filter cap Hand tighten the trap and cap until they are fully closed A To prevent over pressurization during fluidics startup do not overtighten the trap or attached filter cap Tighten each component only until it is hand tight Do not use sealants such as Teflon tape or other adhesives 6 If one month has passed since you last changed the cap replace the filter cap with a new one Write the date on the new cap as a reminder Space for date 7 Reconnect the sensor and fluid line connectors to their respective ports Checking Cytometer Performance 116 Before setting up an experiment you should first run a performance check A performance check ensures that the cytometer is performing consistently over time It also generates default cytometer settings that places each PMT within an optimal range
161. ignal troubleshooting 238 folders adding 132 311 forward scatter FSC about 32 detector 40 ND filter 223 257 removing 223 frequency drop drive 47 FSC See forward scatter G Gap 46 48 gating compensation controls 137 data 143 during sorting 161 global worksheets adding sort layouts 89 162 previewing data 138 setting up 140 H hardware ACDU 168 hazard symbols xiv hazards mechanical 25 heating samples 66 100 height parameters 42 holders collection tube 17 152 300 optical filter 257 sample tube 10 Home Device 84 170 171 hydrodynamic focusing 31 Initial mode 58 installing collection tube holders 152 300 nozzle 205 plates 169 302 sample line filter 212 sample tubes 10 slides 169 302 splash shield 168 instrument See cytometer integrated nozzle about 12 changing 205 cleaning 206 replacing seal 208 interrogation point 11 L labels parameter 139 label specific tubes 134 labware parts list 263 laser delay adjusting 310 lasers about 18 configurations 19 266 delay 43 317 diode 23 51 power 24 shutting down 177 181 specifications 251 starting 100 warmup time 100 layout See sort layouts leaks troubleshooting 244 lens strobe 217 Index 331 levels fluid 67 sample 9 lever nozzle 103 205 light detection 34 injection chamber 9 scatter signals 32 voltage warning 15 limitations xvi Link amp Save 137 Load button 82 loader plate 18 loading tubes 10 30 long
162. in and eyes To prevent retinal burns and possible blindness do not remove laser shielding adjust laser controls or attempt to service the cytometer any place where laser warning labels are attached See the BD FACSAria II Safety and Limitations Guide for the placement of laser warning labels 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 17 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 Chapter 1 Cytometer Components 19 Figure 1 17 Excitation optics pathway behind laser shielding Fiber optics Focusing lens Upper camera Collection Optics From the cuvette flow cell laser light is collected by a fluorescence 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 Figure 1 18 20 BD FACSAria II User s Guide Figure 1 18 Side view of flow cell nozzle and objective lens Stream Cuvette flow cell Individual collection fibers g
163. inches 12 7 cm of tubing extending out of the sample injection chamber end This length can be adjusted depending on the depth of your sample tube This length is referred to as the pilot as shown in Figure 6 16 on page 198 Figure 6 16 Components of a compression fitting External view Internal view Ferrule a Pilot length Tubing Pilot Tubing Pilot depth length 7 Insert the pilot tubing into its fitting ensuring that the tubing reaches the intended pilot depth A Do not to bend the primary sample line during insertion 198 BD FACSAria II User s Guide Insert the sample line into the sample injection chamber fitting Push the tubing from the top until it is slightly above the bottom of the chamber viewing window Finger tighten the nut on top of the chamber so the sample line is secure A Do not overtighten the nut and do not use tools Over tightening the nut can kink or damage the tubing 8 Check the fitting connections at both ends to make sure they are not leaking Turn on the stream load a tube of DI water and make sure none of the fittings are leaking If needed unload the tube turn off the stream and tighten the fittings After tightening if leaking still occurs replace the ferrule A A Make sure all fittings are securely tightened If any fitting is loose the MEX tubing could detach during high pressure operation exposing the operator to potentially biohazardous sample spray 9 Verify the l
164. ine Backflush p Click Start to start the backflush click Stop or Cancel when 2 the backflush is complete 3 Click Stop to stop the backflush or click Cancel to stop the backflush and close the dialog The backflush does not stop automatically Prime After Tank Refill Use the Prime After Tank Refill command to prime the fluid lines if a 5 liter plastic fluidics container was disconnected for refilling 1 Turn off the stream 2 Select Cytometer gt Cleaning Modes gt Prime After Tank Refill 184 BD FACSAria II User s Guide 3 Select the checkboxes for the tanks that were refilled then click OK Tank Prime Please select the checkboxes for the tanks that need to be primed Bleach _ Ethanol The cytometer proceeds with priming the specified tanks A progress message appears while the tanks are being primed Tank Prime Status i Tank prime in progress Please wait 4 Click OK when the tank prime is complete Tank Prime Status i Tank prime is complete Removing the Sheath Probe The sheath probe must be removed from the sheath tank before autoclaving the tank in preparation for performing the aseptic sort procedure NOTE Do not autoclave the sheath probe It is not designed to withstand the conditions of autoclaving Chapter 6 Shutdown and Maintenance 185 Make sure to follow the steps below in sequence so the containment device works properly 1 2 D
165. is appendix provides a list of supplies and options that are available for the BD FACSAria II cytometer e To order spare parts and consumables from BD Biosciences from within the US call 877 232 8995 or go to bdbiosciences com Outside the US contact your local BD Biosciences representative e To order cytometer options contact your sales representative This information is correct at the time of publication For up to date information see our website bdbiosciences com e Cytometer Supplies on page 256 e Consumables on page 261 255 Cytometer Supplies Optical Components The following filters and mirrors are mounted on the BD FACSAria II cytometer Use these part numbers if you need to order any replacement components See Using Custom Optical Filters on page 222 for instructions Detector Array LP Mirror Replacement Laser PMT BP Filter Part No Intended Dye Octagon A 735 343787 488 nm blue laser 780 60 343788 PE Cy7 B 655 343789 695 40 343790 PerCP Cy5 5 or PI 675 20 343791 PerCP C 610 640879 616 23 640880 PE Texas Red D 556 343794 585 42 343796 PE or PI E 502 343797 530 30 343798 FITC F 488 10 343799 SSC 1 0 ND 53 10057 01 filter Trigon A 735 343787 633 nm red laser 780 60 343788 APC Cy7 B 660 20 343800 APC 256 BD FACSAria II User s Guide The following filters and mirrors are provided with the violet laser option
166. isconnect the air line from the sheath tank Vent the air pressure from the sheath tank by pulling up on the pressure relief valve Verify that all of the pressure is released by pulling up a second time Loosen the nut at the top of the probe with an 11 16 inch wrench provided in accessory kit See Figure 6 3 Figure 6 3 Sheath probe containment device Sheath probe Thumbscrew 11 16 inch nut Containment device Loosen the thumbscrew on the containment device Pull the top section of the containment device straight up and out of the bottom section See Figure 6 4 Figure 6 4 Removing sheath probe Top section of containment device 186 BD FACSAria II User s Guide Finish loosening the 11 16 inch nut at the top of the probe and pull the probe straight up and out of the sheath tank Decontaminate the sheath probe using 70 ethanol See Figure 6 5 Figure 6 5 Sheath probe B Prepare for Aseptic Sort Use the Prepare for Aseptic Sort command when you want to decontaminate the entire sheath path This procedure cleans the system with bleach DI water and ethanol Do these steps before starting the Prepare for Aseptic Sort command 1 Verify that the pressure has been vented from the sheath tank and the sheath probe has been removed See the preceding section Disconnect the fluid and air lines from the sheath tank Empty the sheath tank and rinse with DI water Autoclave the sheath tank at 12
167. ishing the aseptic sort prep process 68 To continue running samples perform fluidics startup D To turn off system perform flow cell cleaning procedure e To continue running samples perform fluidics startup e To turn off the system perform the flow cell cleaning procedure Purging the Fluid Filters Once a week purge air from the fluid filters for the 5 L plastic containers by opening the bleeder valve on the top of each filter This ensures that the filters will not dry out 1 Open the bleeder valve a small amount and leave it open until fluid seeps out through the valve see Figure 6 9 on page 191 2 Close the valve 3 Wipe up any excess fluid that might have dripped onto the fluidics cart 190 BD FACSAria II User s Guide Purging the Sheath Filter Once a week purge air from the sheath filter by opening the bleeder valve on the top of the filter The sheath tank is pressurized so do this task carefully to avoid spraying sheath fluid on any equipment 1 Place a small container under the bleeder valve to catch any fluid 2 Slowly open the bleeder valve a small amount and leave it open until fluid seeps out through the valve see Figure 6 10 on page 192 3 Close the valve 4 Wipe up any excess fluid that might have dripped onto the fluidics cart Changing the Fluid Filters We recommend changing the fluid filters every 6 months Spare filters are included with the accessory kit Figure 6 9 Fluid filters
168. l on the diode laser Figure 1 21 on page 23 to better view the streams Select Sort gt Home Device In the Device Setup dialog select the collection device you are using and click Go to Home Device Setup 16 Well Falcon Name 24 Well Falcon Home Pos 925 7565 Go to Home Set Home 3 Falcon Slide Frosted End Slide Standard Test sort button Chapter S Sorting 171 10 11 12 13 The stage moves to the pre programmed home position Double click the Test Sort button to deposit a drop at the home location Inspect the collection device to see where the drop was deposited If you need to move the stage to the front close the Device Setup dialog and click the Access Stage button in the Sort Layout window Wipe the collection device dry and place it back on the tray support If needed click the Access Stage button to send the stage back and select 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 button to turn off the deflection plates Proceed with Sorting on page 161 Creating a Custom Device You can program the ACDU stage to sort into any grid configura
169. late containing small volume of DMEM containing 10 FBS and 10 mmol HEPES Keep sample chilled at 4 C Mi Tip Sort rare event samples into polypropylene tubes Avoid polystyrene tubes Figure B 5 Typical plot from side population experiment Mu BM P1 1 000 ajo Hoechsst 450 50 4 150 290 TE p pi ap p e e e 260 Hoechst 670 LP A amp 1 000 Appendix B Near UV Laser Option 273 Troubleshooting Observation Signals dim or noisy Possible Causes Wrong laser turned on Near UV instead of violet or vice versa Recommended Solutions Turn laser power switch to correct position Laser delay set incorrectly possible holdover from setting on other laser Recalibrate laser delay No signal Laser shutter closed presumably because flow cell access door is open Close flow cell access door Laser power switch set to OFF Turn switch to Near UV Laser power switch set to violet laser instead of Near UV Turn switch to Near UV No signals for side population application Wrong filters installed Install filters for side population application 274 BD FACSAria II User s Guide Appendix C BD Aerosol Management Option The BD aerosol management option AMO is a device that uses an attached vacuum source to rapidly evacuate aerosolized particles through an ultra low penetrating air filter during routine sorting or ana
170. lean the integrated nozzle 1 2 6 7 Turn off the stream and open the flow cell access door Remove the nozzle from the cuvette flow cell Turn the nozzle locking lever counter clockwise to the 6 00 position See Figure 6 20 on page 205 Remove the nozzle by pulling it straight out Caution Do not open the sort block door while the nozzle locking lever is in the 6 00 position The flow cell can be damaged by the top of the door during opening Always turn the lever clockwise back to the 12 00 position before opening the sort block door Sonicate the nozzle for approximately 1 minute Sonicate the nozzle in a test tube containing DI water Repeat the sonication in one minute intervals until the nozzle is clean Do not sonicate for more than a total of 5 minutes Do not use bleach or any strong detergents to clean the nozzle These agents can damage the seal in the integrated nozzle Carefully reinsert the nozzle into the flow cell and push it gently all the way forward until it stops Turn the nozzle locking lever clockwise to the 12 00 position Turn on the stream and make sure it flows through the nozzle properly Close the flow cell access door Note that after re installing 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 both sides of the deflection plates and rotate the sort block see Figure 1 12 on page 14 Tighten
171. lection chamber door and one in the side door e To change the filter in the sort collection chamber door slide out the old filter and slide in the new filter See Replacing the Air Filter on page 288 for more information e To change the air filter in the side door Figure 6 18 loosen the screws at the top corners of the filter and remove the old filter Install a new filter and tighten the screws to hold the filter in place Replace the filter once or twice a year depending on the quality of the air in the laboratory Figure 6 18 Changing the air filter Air filter in right hand side door 202 BD FACSAria II User s Guide Changing the Sheath Tank Air Filter Check the inline air filter on the sheath tank air line periodically for any signs of debris or discoloration Replace with a new air filter from the accessory kit every six months or sooner if needed Figure 6 19 Inline air filter for sheath tank Sheath tank inline air filter 1 Turn off the cytometer 2 Pull the tubing off each end of the air filter 3 Install a new filter with the directional arrow pointing toward the sheath tank Checking the Fluidics Cart Drip Tray Check the drip tray on the fluidics cart weekly for signs of excess liquid and empty the tray if needed Drip tray Chapter 6 Shutdown and Maintenance 203 Unscheduled Maintenance There are several cytometer components that should be cleaned periodically or checked for wear an
172. led In and Out Attach the input tubing to the port on the left side of the collection tube holder and the output tubing to the port on the right side as shown in Figure D 4 on page 300 b To attach the tubing push it into the port until the tubing snaps into place Appendix D Temperature Control Option 299 Figure D 4 Setting up the temperature control tube holder Input tubing Output tubing Tubing ports If you need to remove the tubing push in the orange collar as you pull the tubing out of the port 3 Install the tube holder on the instrument Remove the current tube holder if one is installed and slide the temperature control tube holder into the slotted fittings below the sort aspirator drawer Push the tube holder all the way in 4 Close the sort collection chamber door and start up the water bath 300 BD FACSAria II User s Guide Setting Up the ACDU Stage This section describes how to attach the recirculating water tubing to the stage used with the automated cell deposition unit ACDU A 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 a 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 cham
173. leshooting continued Observation Center stream is off center when the plate voltage is turned on Possible Causes Voltage center too low or too high Recommended Solutions Adjust the Voltage Center slider to put the center stream back to center Saline spray on deflection plates or in sort block Clean the deflection plates and the area around them Arcing between deflection plates Salt bridge Clean and dry the deflection plates and the area around and behind the plates ACDU sorting failure Insufficient stream voltage Increase the voltage for the far left stream Splash shield not installed Install the splash shield See Installing the Sorting Hardware on page 168 Side stream position reversal where the streams appear to be associated with the wrong voltage slider Voltage sliders are set too far in or too far out Move sliders in or out so they control the correct side streams Chapter 7 Troubleshooting 233 Sorting Troubleshooting continued Observation Possible Causes No deflection or Insufficient voltage insufficient deflection Recommended Solutions e Increase the side stream voltages using the slider controls e Increase the plate voltage Stream charging wire is loose or missing Verify the stream charging wire is inserted all the way into the barb Salt bridge Turn off the stream Remove the nozzle See Changing the Integrat
174. lick Done See Figure 6 1 178 BD FACSAria II User s Guide If you are using a standard closed loop nozzle verify that there is an O ring in the nozzle before installing it Figure 6 1 Closed loop nozzle installed in flow cell Connect the air and fluid lines to the stainless steel ethanol ETOH shutdown tank See Figure 6 2 on page 180 a Disconnect the air line from the sheath tank and connect it to the air port on the ETOH shutdown tank b Disconnect the fluid line from the bottom side of the sheath filter and connect it to the ethanol filter on the ETOH shutdown tank See Figure 6 2 on page 180 Keep the sheath filter attached to the sheath tank Do not run ethanol through the sheath filter c Click Done The system starts the cleaning process and then displays a message at the bottom of the window Chapter 6 Shutdown and Maintenance 179 Figure 6 2 Connecting air and fluid lines for shutdown procedure Fluid and air lines moved for shutdown eer Gee Top view Side view y M l Air line Sheath Fluid line filter Ethanol filter _ Disconnect here Ethanol shutdown Sheath tank tank iy ft 5 When prompted install a tube containing 3 mL of sterile filtered DI water on the loading port then click Done Ga Install a tube of cleaning solution onto the loading port The cytometer loads the tube and continues the cleaning process A progres
175. ll it straight out from the slot The tubing pops out of the slot with a small amount of pulling action See Figure 6 29 on page 215 Figure 6 29 Tubing removed from pinch valve Pinch valve collet fittings Sample line tubing Pinch valve tubing P Unscrew the nut on the black collet fitting at each end of the tubing and pull the pinch valve tubing out of both fittings Chapter 6 Shutdown and Maintenance 215 Leave both pieces of the fitting on the sample line tubing 4 Install a new 3 inch piece of pinch valve tubing See Figure 6 30 on page 216 a Slip the collet over one end of the new pinch valve tubing b Slide the pinch valve tubing over the sample line tubing until approximately 6 mm of the sample line is inside the pinch valve tubing c Slide the pinch valve tubing on the inside of the teeth of the nut until it stops d Couple both pieces of the fitting together and then tighten until finger tight e Repeat steps a through d at the other end of the tubing f Check to see that both ends of the new pinch valve tubing are held securely in the compression fittings Figure 6 30 Collet nut fitting on pinch valve tubing Collet Nut Pinch valve tubing Sample line tubing A WZ a hy 6mm 5 Install the new pinch valve tubing into the slot in the pinch valve Make sure the tubing goes all the way into the back of the
176. ltage for that parameter until the positive population can be seen entirely on scale Unload the multicolor sample from the cytometer Chapter 4 Running Samples 129 Saving Application Settings 1 Right click Cytometer Settings in the Browser then select Application Settings gt Save to save the values for reuse See Figure 4 20 Figure 4 20 Saving application settings Save Application Settings Please enter a name 3 color Application Settings a Inthe Applications Settings dialog rename the application settings with a descriptive name b Click OK The application settings are saved to the catalog 130 BD FACSAria II User s Guide Data Collection Before you record data for a sample cytometer settings should be optimized to position the cells of interest on scale for scatter and fluorescence parameters In the previous section application settings were created by taking into consideration the following e FSC and fluorescence area scaling e FSC and SSC voltages e FSC threshold e Fluorescence PMT voltages In this section the application settings will be applied and compensation will be calculated before collecting test data The following sections describe how to use previously optimized application settings for a 4 color experiment See Application Settings on page 123 for more information Compensation will be automatically calculated using the compensation setup feature For more information
177. lters can be changed as often as required The sample filters are not intended for use with the 1 mL microtubes NOTE When a sample filter is installed the sample flow rate can be slowed down due to the effect of particles in the sample fluid clogging the filter 1 Select Change Sample Filter from the Cytometer menu Make sure the stream is turned on before starting the process 2 A wizard appears with the instructions See Figure 6 24 Figure 6 24 Sample line filter wizard D Loosen Pe sample Ine fring on tp of he bulk injecton chamber Ogata Instalvremove he sample titer Raise the samoie line A Place a tube oro the loading pon Cauton Bulk injecton chamber wil close z PO Ea 3 Loosen the sample line fitting nut at the top of the injection chamber to allow the sample line to slide freely through the fitting See Figure 6 25 on page 213 212 BD FACSAria II User s Guide Figure 6 25 Loosen nut to move sample line Sample line Sample injection N chamber Push the sample line down so the end is below the bottom of the sample injection chamber Figure 6 26 Sample line filter installed Sample line filter Install the sample line filter by sliding it onto the end of the line then click Done See Figure 6 26 Do not bend the sample line while installing the filter Pull the sample line up to operation height slightly above the chamber viewing window Place a tube on
178. lysis The following topics are described in this guide e Option Components on page 276 e Operating the BD Aerosol Management Option on page 278 e Maintenance on page 283 e Troubleshooting on page 290 e Specifications on page 293 275 Option Components The BD Aerosol Management Option AMO includes the following e An evacuator to generate negative pressure e An ultra low penetrating air ULPA filter to trap particles with attached tubing that connects the evacuator to the instrument e An air filter for the sort collection chamber door e lt A hinged cover on the sample injection chamber The AMO also requires the splash shield in the BD FACSAria II accessory kit A The BD Aerosol Management Option does not eliminate the health risks of working with biohazardous material and must be used in conjunction with good laboratory practice The BD Aerosol Management Option is For Research Use Only It is not for use in diagnostic or therapeutic procedures Evacuator The evacuator holds the ULPA filter and attached tubing Air flow is controlled using pushbuttons within a membrane panel on the front of the unit The evacuator sits on castor wheels for easy maneuverability It can be moved using the handle attached to the unit See Figure C 1 on page 277 276 BD FACSAria II User s Guide Figure C 1 AMO evacuator 7 Tubin B l Filter hold down ee iy r ULPA filter Membrane A J panel ULPA Filter
179. ment with appropriate tubes plots and labels for your assay This section describes how to add Browser and worksheet elements to the experiment that was started in the previous section Data Collection 1 Rename Specimen_001 to a descriptive name 2 Rename Tube_001 to 4 color_001 3 Set the current tube pointer to the 4 color_001 tube 4 Click Next Tube in the Acquisition Dashboard to duplicate the first tube with the name 4 color_002 5 Use the Experiment Layout to specify the number of events to record for each tube The Experiment Layout can be used to define the events to record as well as parameter labels for all tubes in an experiment Labels appear on the plot axes and in all statistics views a Select Experiment gt Experiment Layout b In the Acquisition tab select the events to record field for all specimen tubes and select or enter 5 000 events c Click OK Chapter 4 Running Samples 139 Experiment Layout Labels Keywords Acquisition Quick Entry Events to Record 5 000 Stopping Gate Stopping Time 1 Global Worksheet Storage Gate Name Events to Record Global Worksh r LJ 4 way calibrate sort dp d GN Analysis Sample d H 4 color_001 DD Gating d U 4 color_002 DD Gating eN Compensation Controls r Ih Unstained Control 5 000 e H FITC Stained Control 5 000 r GPE Stained Control 5 000 e B PerCP Cy5 5 Stained Contre 5 000 e IH APC Stained Control
180. mplitude to maintain the stability of the breakoff point When the Sweet Spot is on the Amplitude and Frequency fields are disabled The values are automatically adjusted by the software For more information see Breakoff Window on page 46 Target value See Table 4 1 on page 107 for examples of breakoff patterns at each nozzle size 106 BD FACSAria II User s Guide NOTE The breakoff patterns in Table 4 1 are intended as examples The patterns will not always look exactly like these Table 4 1 Examples of breakoff patterns at each nozzle size 70 micron 85 micron 100 micron 130 micron FA 70 micron X FA 85 micron X FA 100 mic X fA 130 mic X Stream 2l Stream Stream Al Stream B Sweet Spot Z Sweet Spot B Sweet Spot B Sweet Spot gt 6 6 0 0 103 103 Gling Chapter 4 Running Samples 107 Setting Up the Fluidics Cart Check the fluid levels in the sheath tank and waste containers every time you use the cytometer This ensures that you will not run out of sheath fluid during an experiment or have to service the containers during a sort Fluidics level indicators are shown in the Cytometer window in BD FACSDiva software See Figure 4 2 on page 101 NOTE When the fluidics cart is in the correct position in the lab lock the rolling casters by turning the orange disk until the feet contact the floor This helps minimize any vibrations that c
181. mporary Replacement of a Seal The standard O ring can be used as a short term replacement in the integrated nozzle if the original seal has been lost or damaged but a new integrated nozzle will provide better long term usability See Accessory Kit on page 258 for part numbers for ordering integrated nozzles BD FACSAria II User s Guide Standard O rings part number 333084 are supplied in the accessory kit The following procedure describes how to install a standard O ring in an integrated nozzle 1 Make sure the groove in the nozzle is clean If the any part of the seal is still in the nozzle groove sonicate the nozzle in a 10 bleach solution until the seal comes out Rinse the nozzle in DI water after sonicating Use the wooden end of a cotton swab or similar tool to install the O ring in the nozzle groove then allow the nozzle to air dry for a few minutes Do not wipe the nozzle with anything because it could leave fibers or other contamination or dislodge the O ring Use the magnifier in the accessory kit or a microscope to inspect the nozzle to verify the O ring is installed all the way into in the groove Installing an O ring in a Standard Nozzle The standard O ring can be replaced in the standard nozzle if the original O ring has been lost or damaged Standard O rings are supplied in the accessory kit NOTE We recommend performing this operation under a magnifier due to the small size of the O ring and nozzle
182. mps provide pressure from 5 to 75 psi to accommodate a variety of cell sorting applications Air pressure is adjusted within BD FACSDiva software Containers and Connectors The fluidics cart holds a 10 L stainless steel sheath tank a 5 L stainless steel ethanol shutdown tank a 10 L waste container and three 5 L auxiliary cleaning fluid containers Figure 1 2 2 BD FACSAria II User s Guide Figure 1 2 Fluidics cart containers 3 Auxiliary cleaning fluid containers Sheath filter Waste container Sheath tank Ethanol shutdown tank 3 Fluid filters AA To prevent foaming do not fill the containers with solutions containing a high concentration of detergent The fluidics cart connects directly to the flow cytometer unit via a power cord fluid hoses serial communication cable and air line Figure 1 3 on page 4 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 connecting cables and hoses which extend up to 9 feet 2 7 m Typically the cart is placed to the left or underneath the cytometer Chapter 1 Cytometer Components 3 Figure 1 3 Fluidics cart power and fluid line connectors on cytometer Fluid In Connections for AMO Air In Waste Waste temperature control connection l Air Out Serial option Waste R communication cable Connecting to an External Air
183. n page 134 for a typical error message if there are any mismatches between the application and cytometer settings Chapter 4 Running Samples 133 Figure 4 22 Example mismatch error message Cytometer Settings Mismatch The application settings to be applied do not match the selected cytometer settings The Following parameters are not in the cytometer settings to be applied Cascade Blue A The following parameters are not in the selected application settings FSC H SSC H PE Texas Red A PE Cy7 4 APC Cy A Alexa Fluor 405 4 Alexa Fluor 430 4 Click Apply to apply PMT Voltage and Threshold values only for matching parameters Click Overwrite to replace all parameters and values with those From the selected application settings 10 Select Experiment gt Compensation Setup gt Create Compensation Controls The Create Compensation Controls dialog appears listing only those parameters associated with the application settings 11 Click OK to add the specified controls Alternatively add and define label specific controls then click OK Create Compensation Controls Include separate unstained control tube well Fluorophore e FITC Generic f e PE Generic e PerCP Cy5 5 Generic r APC Generic Add label specific controls when your experiment contains samples stained with the same fluorophore conjugated to different antibodies labels that require different compensation values This is especially us
184. n the bleeder valve on top of the filter a small amount and leave it open until fluid seeps out through the valve 6 Close the valve Changing the Sample Lines The primary sample line between the sample injection chamber and the pinch 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 pinch valve and the cuvette flow cell needs changing only when it is kinked or clogged Figure 6 12 Primary and secondary sample lines Secondary sample line Primary sample line Connecting nut Connecting nut Flow cell assembly ae Sample injection chamber 194 BD FACSAria II User s Guide To withstand the high pressures generated by the BD FACSAria II flow cytometer the sample lines are attached 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 12 inch length of replacement tubing for the primary sample line or a 7 inch length for the secondary line Replacement tubing is supplied in the accessory kit The ferrules and connecting nuts can be reused when the tubing is replaced A All biological specimens and materials coming into contact with them can SX transmit potentially fatal disease Handle used tubing and fittings as if capable of transmitting infection Wear suitable protec
185. n the main power on the water bath control panel Main power switch 2 Use the up or down arrow keys to set the required temperature NOTE To achieve the required sample temperature you will need to set the water bath temperature slightly higher or lower see Table D 1 These settings might need adjustment depending on the ambient temperature in your laboratory We recommend that you calibrate the water bath for your operating environment Table D 1 Water bath settings for corresponding sample temperature Required Sample Water Bath Setting C Temp C 4 2 37 37 5 42 43 2 Appendix D Temperature Control Option 303 3 Wait at least 30 minutes 115 V model or 60 minutes 110 V model to allow the recirculating water reach the required temperature Maintenance 304 To maintain the recirculating water bath see the documentation provided by the manufacturer Tube Holders Remove the tube holders when you are finished using them and clean them periodically before storage A When you detach the recirculating water tubing from the tube holder any fluid remaining in the tubing can leak into the sort collection chamber To ensure that fluid is aspirated from the sort collection chamber make sure the sort chamber aspirator pump is on do not turn off the instrument main power before you detach the tubing Use caution when handling tubing containing hot water To detach the recirculating water tubing pus
186. n the tank is low If the cytometer is run until the sheath supply is too low the system will turn off the stream Figure 4 4 Sheath tank connectors Top view Fluid line Containment device Pressure relief Sheath sensor Fluid connector connector Cover knob Sheath tank Side view Sheath filter To refill the sheath tank 1 Turn off the stream 2 Disconnect the air line 3 Pull up on the ring of the pressure relief valve to release pressure from the tank Make sure the tank is fully vented Chapter 4 Running Samples 109 4 Unscrew the sheath tank cover knob and remove the cover 5 Fill the tank with sheath fluid up to the upper weld line on the inside of the tank See Figure 4 5 NOTE Do not overfill the sheath tank because this can cause incorrect sample flow rates Figure 4 5 Sheath tank fill level Upper weld line to indicate fill level 6 Replace the cover and tighten the knob Make sure the large O ring on the inside lip of the cover is seated correctly and has not slipped out of position The tank can leak if the cover is not secured properly 7 Ifyou removed the sheath tank from the fluidics cart to refill it always place the tank back in its original position on the cart See Figure 4 6 on page 111 The flow rate is calibrated with the sheath tank on the fluidics cart If the location or elevation of the sheath tank is chang
187. ng 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 High sort conflict rate Event rate is too high for drop drive frequency Decrease the event rate Gating conflict Verify the gating hierarchy Purity mask is too high Decrease the purity mask Chapter 7 Troubleshooting 235 Sorting Troubleshooting continued Observation Erratic sort rate Possible Causes Flow rate is too high Recommended Solutions Decrease the flow rate Unexpected sort results Incorrect drop delay Reset the drop delay See Determining the Drop Delay Manual Method on page 154 Incorrect sort precision mode Verify that the sort precision mode is appropriate for your sorting requirements See Sort Precision Modes on page 57 Sweet Spot is 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 Manual Method on page 154 Laser delay changed after setting drop delay Reset the drop delay each time you change the laser delay See Determining the Drop Delay Manual Method on page 154 Incorrect logic in population hierarchy Verify the
188. ng 111 Index 329 events not showing in plots 237 239 rate troubleshooting 240 241 target 90 162 troubleshooting 242 excitation optics 18 251 Experiment Layout 139 experiments Accudrop 97 155 creating 311 cytometer QC 97 doublet discrimination 97 QC setting up 311 reusing 322 setting up 139 sorting 162 templates 97 exporting sort reports 96 165 F FACSAria II See BD FACSAria II FACSDiva software See software ferrules removing 198 200 fiber optics 19 filling containers 112 filters about 35 air changing 202 bandpass 37 changing 191 222 default setup 252 discriminating 37 fluid changing 191 holders 257 longpass 36 neutral density ND 39 223 257 optical 35 223 268 purging 190 removing 191 223 330 BD FACSAria II User s Guide sample line 212 213 sheath 192 Fine Tune mode 58 flow cell about 11 access door 7 cleaning 176 flow rate about 31 adjusting 30 82 recommendations 31 fluid bleach line 189 containers 2 filters 191 level indicators 67 line 102 180 movement 28 out port 189 priming 113 184 recommended 108 refilling 112 fluidics about 28 cleaning 187 components 7 connections 3 containers 2 controls 63 daily shutdown 176 level indicators 67 power 5 shutdown 64 178 startup 29 63 102 system 28 troubleshooting 244 fluidics cart about cord 3 5 dimensions 254 drip tray 203 power 3 5 setting up 108 specifications 254 fluorescence about 33 emission spectra 33 s
189. ng sections Change Sample Filter Cleaning Modes b e Fluidics Controls on page 63 Sheath Pressure Sample Agitation b v a Sample Temperature e Cytometer Configuration on page 68 EENET view Configurations e Cytometer Status Report on page 72 CST Performance Tracking LJ The Cytometer Details and the Catalogs menu commands are described in the BD FACSDiva Software Reference Manual Additional cytometer controls are located in the Acquisition Dashboard See Acquisition Controls on page 81 Catalogs Standby Fluidics Controls Fluidics control of the BD FACSAria II cytometer is partially automated by BD FACSDiva software The software contains pre programmed fluidics protocols that are activated by selecting the corresponding menu command from the Cytometer menu In addition fluidics level indicators are available in the Cytometer window See Fluidics Level Indicators on page 67 Fluidics Startup During fluidics startup waste and sheath levels are verified and the fluidics system is primed with sheath solution The fluidics status is displayed at the bottom of the main window 4 01 25 16 Connected Fluidics Startup done See Cytometer Startup on page 100 for more information Chapter 3 Using BD FACSDiva Software 63 64 Fluidics Shutdown Fluidics shutdown removes sheath fluid from the lines replaces it with ethanol and cleans the cuvette with a cleaning solution See Fluidics Shutdown on p
190. not functioning properly If this occurs contact your BD Biosciences service representative for assistance Do not operate the cytometer outside the normal air pressure range Figure 1 5 Fluidics cart flow gauge Pressure gauge at approximately 88 psi Chapter 1 Cytometer Components 5 See the following sections for more information about the fluidics cart e Setting Up the Fluidics Cart on page 108 e Refilling the Plastic Containers on page 112 e Emptying the Waste Container on page 114 e Scheduled Maintenance on page 182 e Fluidics Troubleshooting on page 244 Flow Cytometer The benchtop flow cytometer contains the major components for all three subsystems fluidics optics and electronics The BD FACSAria II cytometer is relatively compact with a much smaller footprint than most sorters with the same capabilities The cytometer can be set up on a typical laboratory benchtop or table and it requires only a 20 amp electrical outlet No special facilities are required Figure 1 6 BD FACSAria II flow cytometer Flow cell access door Optics access door Sort collection chamber Side door Sample injection chamber 6 BD FACSAria II User s Guide 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 cytometer Other electronic components are embedded within t
191. nt and find the area scaling values in the Tube tab Type these numbers into the Cytometer window Laser tab 322 BD FACSAria II User s Guide Inspector 01Mar07 Tube tab j abels Acq Cytometer Settings Keywords Name O1Mar07 Global Worksheet Total of Events 10 000 Record Date 3 1 07 Record Start 5 43 43 PM Record End 5 44 17 PM Record User Administrator Institution Cytometer Name FACSAria Cytometer Serial 1 Laser Delay Blue 0 00 Red 39 93 Violet 39 87 Area Scaling Blue 1 46 Red 1 36 Violet 1 35 j FSC Area Scaling 1 11 Window Extension 2 00 Area scaling value d Ensure that the red and violet or Near UV laser delays are set appropriately by setting the window extension to 0 and verifying that the area signal is not lost Adjust the laser delay if necessary e Reset the window extension to the appropriate value before verifying the remaining area scaling factors f Verify the red and violet laser area scaling factors Set the flow rate to 1 0 Record 10 000 events Do not adjust any voltages You may need to adjust the singlet gate to capture the population in the FSC A vs SSC A dot plot Appendix E OC Using BD FACSDiva Software 323 Tracking QC Results Monitoring cytometer QC results can provide you with information regarding your instrument s performance Changes in mean fluorescence intensity or CV can alert you to possible instrument i
192. o the worksheet 6 Right click the statistics view and select Edit Statistics View 7 Edit the statistics view as follows e In the Header tab select the Use 2 columns for display checkbox e In the Populations tab clear the checkboxes for all populations except FITC PE PerCP Cy5 5 and APC e In the Statistics tab select the Mean checkboxes for the fluorescence A parameters 8 Resize the statistics view so it fits on the page 9 Optional Print the analysis 144 BD FACSAria II User s Guide Figure 4 27 Sample analysis for mixed bead tube 4 color_001 All Events 4 color_001 FSC Gate sa g 3 ies x 2 3 zg 2 2 a on S Scatter Gate oe a 60 100 150 200 260 50 100 150 200 260 FSC A 1 000 SSC H x 1 000 4 color_001 FSC Gate a 2 O a iva 100 160 200 250 FSC Gate 100 150 200 250 FSC H x 1 000 Tube 4 color_001 Analysis Sample 4 color_001 Population Events Parent Total BAI Events 10 000 AH 100 0 m z BB Scatter Gate 9 341 93 4 93 4 s SSc Gate 9 337 1000 934 FSC Gate 9 336 1000 934 mi PE 228 24 23 E a2 0 0 0 0 0 on E a3 8 903 95 4 89 0 W Fitc 205 22 24 3 E apc 240 2 6 2 4 vor ag wt we fl PercP cy5 5 172 1 8 17 FITC A Analysis Sample 4 color_001 Specimen Name Analysis Sample Experiment Name 4 color Experim Tube Name Record Date 4 color_001 Feb 21 2007
193. of Operation 47 Table 2 1 Breakoff window controls continued Control Description Frequency field In general the drop drive frequency should not need adjustment We continued recommend using the default values that are entered 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 enter a value and turn on the Sweet Spot the cytometer 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 the target value and the actual value is acceptable Gap field The gap between the stream breakoff and the top of the first drop The default Gap setting for a 70 micron nozzle is 6 pixels 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 i
194. old channel Event rate is too high Decrease the flow rate in the Acquisition Dashboard Sample is aggregated Filter the sample Sample is too concentrated Dilute the sample Fewer events than expected in gated population Window extension set incorrectly Adjust the window extension if needed See the BD FACSDiva Software Reference Manual for information Laser delay set incorrectly Adjust the laser delay settings See Cytometer Quality Control Using BD FACSDiva Software on page 308 Plot is zoomed Unzoom the plot or make the gate bigger Events left out of the gate When drawing a gate make sure events on the axis are included 242 BD FACSAria II User s Guide Acquisition Troubleshooting continued Observation Possible Causes Recommended Solutions Increasing threshold Window extension is too low _ Slightly increase the window results in decreased area extension to maximize the area signal signal Increasing the window extension too much results in more electronic aborts or high CVs Area measurement off Area scaling is too high Decrease area scaling to move the scale while the height area measurement back on scale If measurement is on scale necessary adjust area scaling to make the area measurement match the height measurement Cannot delete from Row not selected Select the row using the selection Parameters Threshold button Compensation or R
195. om a sort location field select the field then select the corresponding population from the Delete menu e To clear all populations from a field select the field then select Clear All Chapter 3 Using BD FACSDiva Software 91 92 Using Sorting Controls Sorting controls appear at the bottom of the Sort Layout window Use these controls for the following functions Casa ae Access Stage Move Drawer Pause Resume 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 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 Pause Resume Stops sorting but not acquisition Sort counters and sort timers freeze when the Pause button is clicked Click the Resume button to continue sorting and to continue incrementing the sort counters and timers See Pausing and Resuming a Sort on page 167 Access Stage When the ACDU stage is in the back the stage is moved forward to put a plate on or take a plate off the stage When the stage is in the front the stage is moved to the back and out of the way so you can install a collection tube holder Move Drawer Moves the aspirator drawer in closed or out open depending on its current state The de
196. on 643940 e 85 micron 643941 e 100 micron 643942 e 130 micron not included can be ordered as an 643943 option 644395 e Closed loop Nozzle locking lever spring and plunger included 643686 Standard nozzle O rings 333084 Magnifying glass 337599 Sample injection tubing 12 inch lengths 641059 Sample injection tubing 7 inch lengths 641475 Ferrule 335108 258 BD FACSAria II User s Guide Item Part No Ferrule removal tool 335690 Collet 19 66455 00 Collet nut 19 66456 00 Pinch valve tubing 641900 Sample line filters 35 micron 643152 Sample line filters 50 micron 643153 Sheath fluid filter also used for ethanol shutdown tank 331394 filter Fluid filter for DI water bleach and ethanol tanks 643687 Air filter inline 3 micron 641913 Disposable waste tank caps pack of 12 338854 Cap for 5 L container 5 335916 Lubricant for O rings 347306 O ring pick tool 331430 Deflection plate removal tool 643197 Allen wrench set 98 10004 00 Wrench 11 16 inch for sheath probe 646889 Adapter tray for microscope slides 335630 Power cords e Main power cord 333694 e Cordset for continental Europe 334140 e Cordset for UK 334141 e Cordset for Australia Asia 334175 Appendix A Supplies and Consumables 259 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 repl
197. on page 131 Install the required collection device and set up the side streams See Setting Up for Bulk Sorting on page 152 or Setting Up for Sorting Into a Plate or Slide on page 168 150 BD FACSAria II User s Guide 10 Calculate the drop delay See Determining the Drop Delay Manual Method on page 154 Use gating tools and subsetting methods to define the population s of interest Examples of gating analyses can be found in Analyzing Data on page 143 and in Getting Started with BD FACSDiva Software Define a sort layout for the tube containing the defined sort populations and proceed with sorting See Sorting on page 161 Chapter 5 Sorting 151 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 168 A A Any cytometer 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 To set up for bulk sorting 1 Install collection tubes in the required collection device Collection tube holders are available for 1 mL microtubes 1 5 mL Eppendorf tubes 12 x 75 mm tubes and 15 mL centrifuge tubes For compatible tubes see Labware on page 263 2 Install the collection tube holder onto the cytometer a Close the sort block door and op
198. one If you are using a standard closed loop nozzle verify that there is an O ring in the nozzle before installing it 3 Remove the fluid line from the DI water port connect the fluid line from the bleach container to the DI water port then click Done See Figure 6 6 Do not disconnect the sensors from either container 188 BD FACSAria II User s Guide Figure 6 6 Moving the bleach fluid line DI water line Bleach line connected disconnected to DI water port DI water Bleach container container Disconnect the bleach fluid line from the DI water port and connect it back to the bleach container port then reconnect the fluid line to the DI water port and click Done Disconnect the fluid line from the sheath tank at the output side of the sheath filter and connect it to the fluid out port on the side of the fluidics cart see Figure 6 7 Click Done The system cleaning takes approximately 20 minutes Figure 6 7 Moving the sheath fluid line Sheath fluid line connected to fluid out port Chapter 6 Shutdown and Maintenance 189 6 Disconnect the fluid line from the fluid out port on the side of the fluidics cart and connect it to a new 0 2 pm sheath filter See Changing the Sheath Filter on page 192 7 Remove the old sheath filter and connect the new 0 2 ym sheath filter to the liquid port of the sterilized sheath tank 8 To complete the process select one of the two options Figure 6 8 Fin
199. onics see Signal Detection on page 34 Power Panel Power to the cytometer lasers and fluidics cart is supplied by a power cord plugged directly into a standard electrical outlet There are no special facilities requirements The main power button turns on the cytometer 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 Figure 1 22 Flow cytometer power panel on 3 laser system Main power Main Power Cytometer Fluidics cart System circuit Blue power cord power cord breaker Vit m Laser power The cytometer circuit breaker is located next to the fluidics cart power cord Figure 1 22 The switch will need to be reset if there is a power surge in the laboratory BD FACSAria II User s Guide Emergency Stop Button The emergency stop button to the right of the loading port Figure 1 23 is a safety feature that can be used to stop the movement of the loading port and ACDU stage The button lights up when a tube is loading to remind you to keep your hands away from the loading port Figure 1 23 Emergency stop button j eo Emergency stop button D 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
200. or high CVs Cytometer settings adjusted incorrectly Optimize the scatter parameters See Calculating Compensation on page 135 Flow rate is too high Decrease the flow rate in the Acquisition Dashboard Window extension is 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 is clogged or dirty Clean the nozzle as described in Changing the Integrated Nozzle on page 205 Flow cell is dirty Clean the flow cell See Cleaning the Flow Cell on page 176 Let DI water sit for 15 minutes before turning on the stream Repeat as needed Poor sample preparation Repeat sample preparation Area scaling is too low Verify area scaling See Adjusting Area Scaling and Laser Delay on page 310 Chapter 7 Troubleshooting 241 Acquisition Troubleshooting continued Observation Excessive amount of debris in plots Possible Causes Threshold channel is too low Recommended Solutions Increase the threshold channel See Calculating Compensation on page 135 Dead cells or debris in sample Examine the sample under a microscope Sample is contaminated Re stain the sample making sure the tube is clean High electronic abort rate gt 10 of system event rate Window extension is too high Decrease the window extension Threshold channel is too low Increase the thresh
201. or for any damages resulting from the application or use of this information BD Biosciences welcomes customer input on corrections and suggestions for improvement BD BD Logo and all other trademarks are property of Becton Dickinson and Company 2009 BD Clorox is a registered trademark of The Clorox Company Fluoresbrite is a registered trademark of Polysciences Inc JDS Uniphase is a trademark of the JDS Uniphase Corporation Microsoft Windows and Excel are registered trademarks of Microsoft Corporation Sapphire is a trademark and Coherent is a registered trademark of Coherent Inc SPHERO is a trademark of Spherotech Inc Texas Red Alexa Fluor and Cascade Blue are registered trademarks and Pacific Blue is a trademark of Molecular Probes Inc Teflon is a registered trademark of E I du Pont de Nemours and Company Contrad is a registered trademark of Decon Labs Inc Point Source and iFlex2000 are trademarks of Point Source Ltd Kimwipes is a registered trademark of Kimberly Clark Corp Lauda is a registered trademark of Brinkman Instruments Inc All other company and product names might be trademarks of the respective companies with which they are associated Cy is a trademark of Amersham Biosciences Corp Cy dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use Any other use
202. orresponding counter is hidden Only counters with a checkmark next to the name are displayed Chapter 5 Sorting 163 Starting and Monitoring the Sort 1 Open the sort collection chamber door and install the collection tubes plate or slide 2 Install the sample tube onto the loading port and click Load 3 Adjust the flow rate Sorting results are typically optimized at lower flow rates 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 appears where you can turn on the plates and open the aspirator drawer by clicking OK 5 Verify that the current tube pointer is indicating the appropriate tube in the Browser then click Sort 6 Click OK if you are prompted to open the aspirator drawer or turn on the deflection plates F Confirm 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 and turn plates ON Click Cancel to start sort in current state A If you click Cancel sorting will begin with the deflection plates off and the drawer closed As a result 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 164 BD
203. ou need to rename the configuration right click the new configuration and select Rename Editing the Copied Configuration Once the base configuration is copied you can customize the parameters filters and mirrors in the configuration 1 In the configuration list right click the new configuration and select Edit Configuration The following window appears Copy of 70 70 PerCP cont 5 Cytometer DO FACSAna Ii Crlumoler Hame FACS Arnall Smii Number 1 Copy of 70 70 PerCP conf 5 Blue Laser Red Laser 633m aree OS amp Violot Laser 407nm Use the tabs at the bottom of the window to view each laser s detectors separately Chapter 3 Using BD FACSDiva Software 77 2 To edit the nozzle size and sheath pressure for this configuration a Enter the appropriate sheath pressure value at the bottom of the configuration window b Select the appropriate nozzle size Sheath Pressure psi 70 00 Window Extension us 20 Nozzle Size p 70 v 3 To edit the optical configuration e To change the parameter label for a detector select and drag a new parameter from the Parameters list to the appropriate detector Parameters FP GFP Hoechst Hoechst 33258 Hoechst Blue Hoechst Red Indo 1 Blue Indo 1 Violet Marina Blue Pacific Blue PE PE Cy7 Ce PerCP Cy5 5 PE Texas Red PI Qdot Qdot 525 Qdot 565 Qdot 585 Qdot 605 Qdot 655 uyi U 2 w gt Select and drag onto t
204. oubleshooting 231 Sorting Troubleshooting Observation Possible Causes Unstable breakoff while Residual ethanol in system Sweet Spot is engaged Recommended Solutions Allow the system to run until the breakoff stabilizes Target Drop 1 value is out of range for drop spacing Use an actual Drop 1 value for the target Remember to repeat the drop delay setup each time the target value is changed Nozzle is clogged or inserted improperly Turn off the stream Remove the nozzle and clean it Re insert the nozzle and slide the nozzle in until it stops then close the locking lever Dirty strobe lens or upper camera window Clean the lens and the window as described in Cleaning the Camera Windows on page 217 Air in sheath filter Purge the sheath filter See Purging the Sheath Filter on page 191 Debris in flow cell or nozzle Remove the nozzle and run the stream with no nozzle in place for approximately 10 seconds Click the Stream control on and then off Sonicate the nozzle and re install it Center stream image is Camera window is dirty dim or not visible in the Side Stream window Clean the lower camera window See Cleaning the Camera Windows on page 217 Stream is not intercepting the diode laser Adjust the micrometer dial on the laser to make the stream intercept as bright as possible See Figure 1 21 on page 23 232 BD FACSAria II User s Guide Sorting Troub
205. ould affect the stability of the stream 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 2 For ordering information see Reagents on page 262 For instructions on emptying the waste see page 114 Table 4 2 Recommended fluids Container Compatible Fluids Capacity Sheath BD FACSFlow sheath fluid non sterile One 10 L e 1X phosphate buffered saline PBS with or without stainless ee container preservatives Ethanol 70 solution diluted in DI or laboratory grade water One 5 L stainless Shutdown steel container Bleach e 10 household bleach 0 5 sodium hypochlorite SL e BD FACSClean solution DI Deionized water Milli Q or equivalent Add 3 mL of SL bleach per liter of DI water Ethanol 70 solution diluted in DI or laboratory grade water SL a Users should select solutions that are compatible with their specific samples and experiments 108 BD FACSAria II User s Guide NOTE Make sure the blue sheath fluid line between the sheath tank and the cytometer does not come into contact with anything that could introduce vibration that might affect the stability of the stream Refilling the Sheath Tank The fluid level in the pressurized stainless steel sheath tank should be checked often and refilled when low The startup times for the breakoff stream will increase whe
206. owing the progress of the sort Figure 3 12 Sort Layout during sorting Global Sheet1 Sort Layout_001 Device Precision Target Events Save Sort Reports Save Conflicts 4 Way Purity l tin Ask User v Far Left Left Right Far Right PE 42547 B Fc 17752 Mi arc 20910 I Percr cy5 18389 Sort Rate Confl Cnt Sort Report Select Sort gt Sort Reports to view all of the saved reports for the current sort layout see Figure 3 13 A sort report can be printed or exported The Sort Reports menu option is enabled if either of the following is true a sort layout is selected in the Browser even if experiment is closed or a sort layout is open If both conditions are true the Browser selection takes precedence BD FACSAria II User s Guide Figure 3 13 Select Sort Reports dialog Select Sort Report Fri Jan 26 09 48 02 PST 2007 Fri Jan 26 09 45 14 PST 2007 A sort report see Figure 3 14 on page 96 contains the following e Header information Experiment name specimen name 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 mask definitions 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
207. owser 2 Cytometer 1 Inspector l Worksheet and Acquisition Dashboard windows as needed Mi Tip As you work in the software windows can become hidden You can easily bring a window to the forefront by double clicking the corresponding button on the Workspace toolbar 2 Create a folder for the QC experiment In the Browser select the icon for your database and click the New Folder button on the Browser toolbar Rename the folder Cytometer QC or create a Cytometer QC folder inside another folder See the BD FACSDiva Software Reference Manual for ideas on how to organize experiments New folder button _ al Fie m E Fi al is HE Nome Database icon Eg Administrator Folder_00 Shared View 3 Create an experiment from the QC Experiment template Select Experiment gt New Experiment click the General tab and select the QC Experiment template Figure E 3 Click OK to add a copy of the template to the Browser Appendix E OC Using BD FACSDiva Software 311 Figure E 3 Creating an experiment from the QC template Experiment Templates a General G5 Practice Name Date Name QC Experiment Blank Experiment l ccudrop Drop Delay 11 22 06 11 00 AM Blank Experiment with Sample Tube 11 22 06 11 00 AM Doublet Discrimination Gating 11 22 06 11 00 AM QC Experiment 11 22 06 11 00 AM Name QC Experiment a ae QC Experiment Copies 1 Cancel
208. ozzle see Cleaning the Integrated Nozzle on page 206 Optional You can print the sort report at this time or open the report later and print it then You can also export the report Chapter 5 Sorting 165 Stopping and Resuming a Sort The Stop Resume feature allows you to temporarily stop the sort and still retain the counter values This is particularly useful when the sample volume is low and you need to refill the tube or to replace collection tubes Replacing Sample Tube 1 To stop a sort while it is running click the Sort button in the Sort Layout window If prompted click OK to save the sort report You can set the sort report to save automatically after each sort See Setting Up a Sort Layout on page 89 Unload the sample tube by clicking Unload in the Acquisition Dashboard Refill the sample tube then click Load in the Acquisition Dashboard Click the Resume button in the Sort Layout window to continue sorting Click OK when you are prompted to open the aspirator drawer or turn on the deflection plates The sort counters resume from the value where they stopped The threshold counter restarts however the value is accumulated and the total count is saved in the final sort report Replacing Collection Tubes 1 To stop a sort while it is running click the Sort button in the Sort Layout window If prompted click OK to save the sort report You can set the sort report to save automatically after each sort
209. pening the Sort Collection Chamber Door 00000 ee eee 282 Turning Off the Evacuator 0 0c ccc cece nent ene 283 Maintenance srias ieena E a aet Paes oe ha aa ee 283 Replacing the ULPA Filter 2 0 0 cece ccc eee eee nes 284 Replacing the Air Filter 2 0 ee ccc eee nen eens 288 Troubleshooting ais fe ocala 40d ae Sele Bai bs Shoes Sac es SREP SS 290 Control Panel Troubleshooting 0000s 290 Filter Flow Gauge Troubleshooting 0 000 cece eee eee 292 Specifications riera eewrite age a T gd etl Sandy boii O 293 Appendix D Temperature Control Option 295 Option Components oers rip TUE eee eee eee eee eee ene e eens 296 Using the BD Temperature Control Option 0 0c cece eee eens 297 Setting Up the Water Bath 0 0 eee ccc eee 297 Setting Up the Tube Holder 0 0 cece ccc eens 299 Setting Up the ACDU Stage cece cc cee een eens 301 Starting Up the Water Bath 0 cee ce eens 303 Maintenance sesruro see Aaa e he late oie ate iw a a eae eas 304 TPubesHOlders o ee gee are eens aE ee ae a Le aie ate dae 304 Recirculating the Water Tubing 0 0 304 Specifications Ae sic seine dade whys a bine Ded wa Gea iad wie es 305 Contents xi Appendix E QC Using BD FACSDiva Software 307 Cytometer Quality Control Using BD FACSDiva Software 000 308 Setting Up the Cytometer Configuration 000 e cece eee eens 308 Preparing QC Particles 0
210. ps to be sorted However because the target particle is closer to the edge of the drop there is more variability in drop trajectory See Figure 2 23 Figure 2 23 Sorted drop with phase mask of 8 Trailing drop Drop sorted Leading drop il M O L L Phase mask Mi Tip We recommend using a phase mask of at least 8 when sorting single cells Phase masks cannot be used in conjunction with yield masks Therefore when the phase mask is greater than zero the yield mask automatically reverts to zero 56 BD FACSAria II User s Guide Sort Precision Modes Mask values can be combined in many different ways By default six sort precision modes are already defined Purity 4 Way Purity Yield Single Cell Initial and Fine Tune amp Sort Precision Precision Mode v Yield Mask 32 Purity Mask 32 Phase Mask 0 Single Cell Precision Mode Purity pl Yield ar Initial a Yield Mask 32 0 32 0 32 0 Purity Mask 32 32 0 32 0 0 Phase Mask 0 0 0 16 0 0 Single Cell O O O O O e Purity mode The yield mask is set to the maximum to obtain the greatest number of particles Because the purity mask is also set to the maximum only drops free of contaminating particles will be sorted Sorting in Purity mode results in a sorted sample that is highly pure at the expense of recovery and yield e 4 Way Purity mode The purity mask is set to the maximum
211. r s Guide e Select the Statistics checkbox to export the statistics to a separate file then enter a name for the statistics file By default the file is saved in D BDExport Statistics e Select the Freeze Biexponential Scales checkbox to process all files with the same biexponential scales e Clear the Use Preferred Global Worksheet checkbox to display analyses of tubes within the same global worksheets This option is useful for analyzing panels that require a separate global worksheet for each tube Click Start to begin the analysis When the analysis is finished a completion message similar to Figure 4 28 displays Figure 4 28 Batch analysis complete Batch Analysis Complete i The batch processing is finished Pages have been printed Statistics output to D BDExport Statistics Batch_Analysis_02032007173318 cs Chapter 4 Running Samples 147 THIS PAGE INTENTIONALLY LEFT BLANK D 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 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
212. r is open Aspirator Drawer The aspirator drawer keeps the sort collection tubes covered until sorting begins Figure 1 13 You can open and close the drawer using a control in the Sort Layout or Side Stream window see Using Sorting Controls on page 92 When the Sweet Spot is on and a clog is detected during sorting the drawer automatically closes to protect the sort collection tubes A To avoid pinching your hands or fingers in the drawer keep your hands away from the sort block during sorting Figure 1 13 Aspirator drawer closed left vs open right Chapter 1 Cytometer Components 15 16 Aerosol Management During sample acquisition and sorting the sort block door and flow cell access door should be kept closed to help contain potential aerosols Figure 1 14 A A Cell sorters that use droplet generation methods like the BD FACSAria II 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 and you are working with highly infectious samples consider turning off the stream before opening the sort block door Figure 1 14 Sort block with door closed Sort block Deflection plates warning light Sort collection chamber Additional aerosol removal is provided by the BD Aerosol Management Option AMO The AMO is an optional
213. rdation and fungi resistance e High dust trapping capacity low air resistance e Can be used in a wide variety of climatic conditions e Rated UL 94 HF 1 Appendix C BD Aerosol Management Option 293 THIS PAGE INTENTIONALLY LEFT BLANK Appendix D Temperature Control Option The BD temperature control option can be used to control the temperature of sorted samples in the BD FACSAria II flow cytometer The following topics are described in this guide e Option Components on page 296 e Using the BD Temperature Control Option on page 297 e Maintenance on page 304 e Specifications on page 305 295 Option Components The BD temperature control option includes the following e A recirculating water bath e Specially designed collection tube holders with ports for recirculating water Tube holders are available in these styles two way 15 mL four way 12 x 75 mm and four way 1 5 mL Eppendorf tubes See Figure D 1 Figure D 1 Temperature control option components Two way 15 mL Four way 12 x 75 mm Four way 1 5 mL Eppendorf Water bath 296 BD FACSAria II User s Guide A When acquiring biohazardous samples follow universal precautions at all times Keep the sort block door and the sort collection chamber door closed during sorting We recommend that you use the BD Aerosol Management Option when sorting biohazardous samples into the temperature control tube holders The BD temperature control option is For
214. re B 5 on page 273 for a typical plot from a side population experiment Instrument Setup 1 2 Turn on Near UV laser and allow it to warm up for 30 minutes Make sure the cytometer configuration is set to a configuration with Near UV parameters Ensure that the side population filter set is in the Near UV far left trigon Place the 670 LP filter Hoechst Red in detector A with 610 LP mirror in front Place the 450 20 filter Hoechst Blue in detector B See Figure B 3 on page 268 Make sure the cytometer has a valid performance check or perform QC Experiment Setup Tips 1 2 Set the parameters to linear in the experiment Create a Hoechst Red vs Hoechst Blue dot plot Add a FSC vs PI dot plot if you want to discriminate dead cells from viable ones Set the sample temperature to 4 C by selecting Cytometer gt Sample Temperature Set the sample flow rate to as low as possible to achieve the best CVs Sample concentration should be between 1 and 2 million cells per mL of buffer 272 BD FACSAria II User s Guide Adjust the voltages so that the red blood cells are seen in the lower left corner and the dead cells line up on the vertical line to the far right The bulk of the rest of the cells can be centered It should be possible to identify a major Go G population with S G Collect a file of 50 000 to 100 000 of live gated events SP prevalence is 0 02 to 0 05 of total If sorting sort into tubes p
215. rking with the system with the Near UV laser installed is familiar with safety details in the BD FACSAria II Safety and Limitations Manual Appendix B Near UV Laser Option 267 Operation Selecting Optical Filters When using the Near UV laser for side population studies you can install a different set of filters in the far left trigon The filter set is supplied with the system when the Near UV option is installed This set is optimized for side population stem cell applications See Table B 2 for the part numbers and intended dyes and Figure B 3 for the locations in the trigon Table B 2 Part numbers for filters for Near UV trigon Detector Array LP Mirror Replacement Laser PMT BP Filter Part No Intended Dye Trigon 375 nm A 610 LP 640879 Hoechst Red PI Near UV laser 670 LP 642328 B 450 20 642329 Hoechst Blue DAPI Pacific Blue Cascade Blue Figure B 3 Trigon with Near UV filters installed for side population studies 268 BD FACSAria II User s Guide Creating a Custom Configuration The Near UV laser uses the same PMT detectors as the violet laser but typically with different filters installed for optimizing side population studies We recommend that you set up a custom configuration for the Near UV laser in BD FACSDiva software using the CS amp T application See Figure B 4 Figure B 4 Three laser system configuration with Near UV laser 3 laser 9 color 5 2 2 Con
216. rning 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 j Fluidics i Waste is full Please empty A TAN not dismiss the warning message without refilling the sheath or emptying the waste If you continue to run the cytometer after dismissing the message the system could shut down Chapter 3 Using BD FACSDiva Software 67 68 Cytometer Configuration The menu selections shown in Figure 3 4 open the Cytometer Setup and Tracking CS amp T module in a separate window The CS amp T module enables you to perform multiple functions related to cytometer configuration NOTE When the CS amp T module is open BD FACSDiva software goes into a holding mode and does not accept any user input When CS amp T is closed BD FACSDiva becomes active again Figure 3 4 Cytometer menu selections that open CS amp T cytometer Sort Help Fluidics Startup Fluidics Shutdown Change Sample Filter Cleaning Modes b Sheath Pressure Sample Agitation Sample Temperature b Cytometer Details view Configurations Menu selections that open the CST CS amp T module Performance Tracking LJ Catalogs Standby e View Configurations Opens the Cytometer Configuration window within the main CS amp T window See the following section for an overview of this function e CST Opens the CSXT module
217. rols The Breakoff window displays an image of the stream and contains controls to adjust drop formation See Drop Formation on page 45 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 49 Sort Menu Select commands in the Sort menu for the following Sort Setup gt e Sort Setup Downloads the most recently used settings por precson i for selected nozzle size See Sort Setup on page 85 z P Home Device e Sort Precision Opens a dialog where you can select or Custori Dewees define a sort precision mode for handling sorting conflicts See Conflict Resolution During Sorting on page 53 e New Sort Layout Opens the default 2 Tube Sort Layout window where other sort layouts can be selected See Sort Layout on page 87 Note that clicking the Sort Layout button on the Browser toolbar performs the same function e 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 e Home Device Opens a dialog containing commands to move the tray support arm either manually or to the home position See Setting Up the Stream on page 170 e Custom Devices Opens a dialog where custom devices can be defined See Creating a Custom Device on page 172 84 BD FACSAria II User
218. roperly Turn off the stream Remove the nozzle and ensure that the seal or O ring is in place Re insert the nozzle and slide the nozzle in until it stops then close the locking lever 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 the Integrated Nozzle on page 206 e If the nozzle appears damaged replace it See Changing the Integrated Nozzle on page 205 226 BD FACSAria II User s Guide Troubleshooting the Stream continued Observation Possible Causes No stream or dripping stream Nozzle inserted improperly Recommended Solutions Turn off the stream Remove the nozzle See Changing the Integrated Nozzle on page 205 for instructions 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 the Integrated Nozzle on page 206 e If the nozzle appears damaged replace it See Changing the Integrated Nozzle on page 205 Stream control disabled Air lock in fluidics filter or no stream when Stream control clicked Prime the system with the corresponding fluid If the control is still 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
219. rt and click OK The cytometer loads the tube and fills the flow cell with the DI water Click OK when the completion dialog appears If this is part of a daily shutdown perform these steps a Turn off the cytometer main power b Exit BD FACSDiva software and shut down the computer c Vent the air pressure from the sheath tank by pulling up on the vent ring Chapter 6 Shutdown and Maintenance 177 Fluidics Shutdown The Fluidics Shutdown command can be used to perform an extensive cleaning if the system is used to process many different sample types or the system has problems with contamination This procedure removes sheath fluid from the lines and fills them with 70 ethanol and cleans the flow cell Preparing for Shutdown 1 Unload the sample tube if one is loaded 2 Turn off the stream 3 Check the waste container and empty it if needed See Refilling the Sheath Tank on page 109 4 Check the ethanol shutdown tank and refill if it needed See Refilling the Ethanol Shutdown Tank on page 111 Running Fluidics Shutdown 1 Select Cytometer gt Fluidics Shutdown The following window opens Fluidics Shutdown D Remove the nozzle from the flow cell 63 Install the closed loop nozzle into the flow cell Zis Connectthe air and fluid lines to the stainless steel ETOH tank 2 Remove the nozzle from the flow cell assembly and click Done 3 Insert the integrated closed loop nozzle into the flow cell assembly and c
220. ry Sample Line Sample Injection End At the sample injection end of the primary sample line a compression fitting secures the sample line at the top of the sample injection chamber Figure 6 14 Primary sample line insertion eee Primary sample line Nut Ferrule Sample injection chamber 1 Turn the stream off if needed Make sure the loading port is in the unload position 2 If there is a sample line filter installed remove it by pulling it off of the sample line To gain access to the filter see Installing or Removing a Sample Line Filter on page 212 3 Unscrew the connecting nut at the top of the sample injection chamber and slowly pull out the sample line See Figure 6 12 on page 194 Chapter 6 Shutdown and Maintenance 197 4 Ensure that a cone shaped ferrule is attached to the sample line Figure 6 14 on page 197 shows an example of a ferrule If the ferrule was left behind in the injection chamber fitting gently push the tip of the ferrule removal tool included in the accessory kit into the top of the ferrule and pull the ferrule straight out See Figure 6 15 After using the tool you might need to replace the ferrule If the ferrule is damaged replace it with a spare included in the accessory kit Figure 6 15 Ferrule tool m MM 5 Slide the ferrule and nut off the end of the sample line 6 Slide the nut and then the ferrule onto the end of the new sample tubing Leave approximately 5
221. s Guide 2 Theory of Operation This chapter describes how the BD FACSAria II cytometer 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 following sections for a description of these BD FACSAria II functions e Fluid Movement on page 28 e Signal Generation on page 32 e Signal Detection on page 34 e Electronic Processing on page 41 e Sorting on page 44 27 Fluid Movement 28 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 II fluidics system Figure 2 1 Fluidic movement via the fluidics system SHEATH FLOW SAMPLE FLOW sheath tank sample tube i sample injection sieht wasteleenirator sample collection P chamber collection tube or plate BD FACSAria II User s Guide Sheath Flow When you turn on the cytometer the main air compressor starts up The fluidics system is activated when you select the Fluidics Startup command in BD FACSDiva software During fluidics startup sheath fluid is forced from the pressurized sheath tank through a filter and is delivered to the cuvette flow cell at a constant pressure You can vie
222. s a valid performance check or perform QC You are now ready to set up and perform your experiment using the Near UV laser 270 BD FACSAria II User s Guide Switching Back to the Violet Laser NOTE Depending on the application the filters for the standard violet laser can also be used with the Near UV laser This section provides an overview of the main steps 1 Place the UV Off Violet laser switch in the Violet position This turns off the Near UV laser and turns on the violet laser Allow the violet laser to warm up for 30 minutes before recording data 2 Set the cytometer configuration to a configuration with violet laser parameters Verify the configuration contains the correct parameters for use with the violet laser The system will not perform correctly if this is not done 3 Install the correct optical filters the violet laser in the far left trigon Detector Array LP Mirror Replacement Laser PRAT BP Filter Part No Intended Dye Trigon A 502 343797 Alexa Fluor 430 405 nm violet laser 530 30 343798 B 450 40 343801 Cascade Blue Pacific Blue Hoechst DAPI Alexa Fluor 405 4 Make sure the cytometer has a valid performance check or perform QC Appendix B Near UV Laser Option 271 Side Population Application Guidelines The Near UV laser on the BD FACSAria II cytometer is very effective for use in applications involving side population SP studies See Figu
223. s message appears and then displays Done when the process is complete 68 Fluidics Shutdown is complete Daily Clean mode in progress Done 100 Cancel 180 BD FACSAria II User s Guide 6 Click OK when you see a message informing you the system can be turned off 7 Vent the air pressure from the sheath tank by pulling up on the ring on the pressure relief valve 8 Turn off the cytometer main power 9 Exit BD FACSDiva software and shut down the computer External Cleaning To keep the system free from salt buildup wipe down all cytometer surfaces that have been exposed to sheath fluid Clean surfaces with a cloth dampened with a 10 bleach solution followed by DI water A A All cytometer surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when cleaning cytometer surfaces Wear suitable protective clothing and gloves AAi 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 chamber e Deflection plates e Sample loading port e Collection devices Chapter 6 Shutdown and Maintenance 181 Scheduled Maintenance For optimal cytometer functioning perform the following procedures according
224. s restored Note that when the sort is paused the sample continues to flow Once the Gap is back within range sorting automatically resumes For information on setting the breakoff see Setting Up the Breakoff on page 105 48 BD FACSAria II 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 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 Drop Delay Overview on page 51 Figure 2 15 Side Stream window FA 70 micron 6 Voltage g Test Sort w Optical Filter El Attenuation E waste Drawer Drop Delay 39 57 a Auto Delay Voltage Center Plate voltage 5 001 ay 2nd Drop 20 Z 3rd Drop 10 4th Drop 0 Phase lt Controls in the Side Stream window are described in Table 2 2 on page 50 You can send different values to the Side Stream window by selecting a nozzle size from the Sort gt Sort Setup menu Note that changes to values in the Sort Setup windows Side Stream and Breakoff are linked to the sort setup and are automatically saved At startup the last settings used on the cytometer are restored except the states of the Voltage Test Sort and Optical Filter buttons which
225. settings determine how drops are deflected when sorting conflicts occur There are three mask Sort Precision i Precision Mode Pri settings each of which addresses a different type of en conflict These settings are combined to define sort ma precision modes Each mode is made up of a set of peste masks Precision modes are defined in the Sort Single Cel Precision dialog accessed from the Sort menu Chapter 2 Theory of Operation 53 54 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 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 Figure 2 18 Target particle within a yield mask of 16 Trailing drop sorted Drop being interrogated Leading drop not sorted A Mi If the yield mask were set to 8 for the same target particle the target particle would fall outside of the yield mask Therefore no additional drops would be sorted See Figure 2 19 a 5 eee Yield mask Yield mask Target particle Figure 2 19 Target particle outside a yield mask of 8 Trailing drop not sor
226. sistance in solving a problem e Read the section of the user s guide specific to the operation you are performing e See Troubleshooting on page 225 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 Details of recent system performance For instrument support from within the US call 877 232 8995 For support from within Canada call 888 259 0187 Customers outside the US and Canada contact your local BD representative or distributor About This Guide XV Limitations xvi This instrument is for Research Use Only Not for use in diagnostic or therapeutic procedures BD Biosciences is providing software without warranty of any kind on an as is basis The software and workstations are intended for running the instruments supplied by BD Biosciences It is the responsibility of the buyer user to ensure that all added electronic files including software and transport media are virus free If the workstation is used for Internet access or purposes other than those specified by BD Biosciences it is the buyer user s responsibility to install and maintain up to date virus protection software BD Biosciences does not make any warranty with respect to the workstation remaining virus free after installation BD Bioscien
227. so only drops free of contaminating particles will be sorted The yield mask is set to zero to ensure that residual charges from adjoining drops do not degrade the quality of side streams The 4 Way Purity mode is recommended for four way sorting where precise deflection is required Chapter 2 Theory of Operation 57 e Yield mode Only the yield mask is used at its maximum value Recovery and yield are optimized at the expense of purity The yield mode could be used as a first round sort for enrichment of target particles followed by a sort for purity e Single Cell mode The purity mask is set to the maximum so only drops free of contaminating particles will be sorted The phase mask is set to 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 This mode is recommended for plate sorting or situations where precise counting is required The remaining modes are used mainly during drop delay determination Initial mode is equivalent to Yield mode but it is named differently as a reminder to use this as the initial mode when using Accudrop to set the drop delay e Initial mode Only the yield mask is used at its maximum value Recovery and yield are optimized at the expense of purity e Fine Tune mode All masks are set to zero to deflect the maximum number of drops This mode is used to fine tune the drop delay value See Determining th
228. ssues or service procedures that may need to be addressed To track cytometer performance over time copy means and CVs for each parameter onto a QC log You can also perform batch analysis which allows you to automatically export a statistics file in comma separated value CSV format for a group of tubes The CSV file can be opened in a spreadsheet application See Performing a Batch Analysis on page 146 for instructions on this process 324 BD FACSAria II User s Guide Index Numerics 4 Way Purity mode 57 A aborts See also conflicts sort electronic 242 Access Stage button 92 accessory kit contents 258 Accudrop about 51 beads sorting in Initial mode 158 experiment 97 155 optimizing drop delay 156 ACDU See automated cell deposition unit acquisition controls 81 events torecord 139 troubleshooting 237 Acquisition Dashboard 81 adding cytometer configurations 74 experiments 311 folders 132 311 sort layouts 89 162 sort populations 90 162 adjusting amplitude 48 105 area scaling 124 126 127 312 Drop 1 48 106 drop delay 156 flow rate 30 82 Home location 170 laser delay 317 micrometer dial 157 160 PMT voltages 42 side streams 170 sort block angle voltages 125 Window Extension 318 aerosol management option AMO about 16 changing filters 202 components 276 connecting 3 maintenance 283 specifications 293 starting up 278 troubleshooting 290 using 275 293 agitating samples 65 air ex
229. stograms and statistics views 126 BD FACSAria II User s Guide 10 11 12 Figure 4 17 FSC area scaling comparison Incorrect FSC area scaling Specimen_001 Tube_ 001 Correct FSC area scaling Specimen_001 Tube_001 ze s a S d 50 100 150 200 250 FSC H k 1 000 Specimen_001 Tube_001 2 22 E 5 a J 3 ol 50 100 150 200 250 FSC A 1 000 Tube Name Tube_001 Record Date FSC A FSC H Population Mean Mean ri 141 366 116 850 EE a oJ 50 100 150 200 250 FSC H x 1 000 Specimen_001 Tube_001 R 2 z as l 50 100 150 200 250 FSC A amp 1 000 Tube Name Tube_001 Record Date FSC A FSC H Population Mean Mean ri 102 830 103 631 Adjust the blue laser area scaling until the FITC A signal matches the FITC H signal if needed Unload the FITC positive control tube then load the APC positive control tube Adjust the red laser area scaling until the APC A signal matches the APC H signal if needed In the Inspector window clear the checkbox for height for all parameters See Figure 4 18 on page 128 Chapter 4 Running Samples 127 Figure 4 18 Deselecting the height parameter Inspector Cytometer Settings Cytometer Settings Parameters Threshold Ratio Compensation Parameter Voltage FSC 186 S5C 306 603 PE 513 597 Optimi
230. t ad a Nozzle SS Nozzle locking lever The unique flow cell design permits particles to flow through the cuvette at a low velocity approximately 6 m sec for the 70 micron Sort Setup allowing longer exposure to laser energy The cuvette is gel coupled to the fluorescence objective lens to transmit the greatest amount of emitted light from the interrogation point to the collection optics see Optics System on page 18 After passing through the cuvette the stream is accelerated to approximately 30 m sec with the 70 micron Sort Setup as it enters the nozzle tip where the drop drive breaks the stream into droplets for sorting Chapter 1 Cytometer Components 11 12 Integrated Nozzle The BD FACSAria II cytometer is provided with integrated nozzles that have the seal fixed into the groove With proper handling and maintenance the seal is designed to stay in the nozzle and not need replacing The integrated nozzles are available in four sizes 70 85 100 and 130 um to accommodate a variety of particle sizes plus a closed loop nozzle for use in cleaning and shutdown procedures The nozzle is keyed to a fixed position at the lower end of the cuvette Because the nozzle is below the interrogation point optical alignment is not affected when the nozzle is changed Ned Figure 1 11 Integrated nozzles Bottom view M indicates integrated nozzle See these sections for more inform
231. t focus each laser on the stream at a separate position This allows optimal detection of fluorescence signals 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 Figure 2 13 Signal separation over time 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 Violet Time gt 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 laser so the red laser signals always have a negative delay value Cytometer FACSAria 1 Status Parameters Threshold Laser Compensation Ratio Name ix Delay Area Scaling Blue 0 00 Red 36 00 36 00 Window Extension 2 00 RH FSC area scalino 100 Sf Cytometer Connected D m a B B Chapter 2 Theory of Operation 43 Sorting 44 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
232. talling 168 stage accessing 92 standard nozzle 208 starting cytometer 100 fluidics 29 63 102 lasers 100 sorting 92 164 stream 104 statistics view creating 144 status cytometer report 72 Stokes shift 33 stop button 25 stopping sorting 92 stream See also side streams centering 50 154 control 47 deflecting 51 flow rate 29 setting up 170 starting 104 troubleshooting 104 226 viewing 23 stream charging wire 53 strobe lens cleaning 217 218 supplies cytometer 256 Sweet Spot about 45 46 control 47 symbols hazard xiv T tanks See containers target events 90 162 technical assistance xv temperature control option about 17 maintenance 304 setting up ACDU 301 specifications 305 using 296 305 water bath 297 temperature sample 66 templates experiment 97 test sort 50 153 171 threshold troubleshooting 243 trap waste 115 trigons about 21 22 34 default filters 252 troubleshooting ACDU 233 acquisition 237 AMO 290 breakoff 105 231 CVs 241 deflection 234 electronic aborts 242 electronics 245 event rate 240 241 fluidics cart 244 leaks 244 low Area signal 239 Near UV laser 274 populations 235 242 pressure 244 scatter parameters 241 signals 238 243 sorting 232 235 236 stream 104 226 threshold 243 tubes adding sort layouts 89 162 agitating 65 compensation 134 heating cooling 66 100 holders 10 17 label specific 134 loading 10 30 replacing 166 sorting into 17 152 unloading 10 30
233. ted Drop being interrogated Leading drop not sorted A M A Vy Yield mask Target particle 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 Ly Yield mask Yield masks cannot be used in conjunction with phase masks Therefore when the yield mask is greater than zero the phase mask automatically reverts to zero BD FACSAria II 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 See Figure 2 20 i Non target particle Figure 2 20 Non target particle within a purity mask of 16 Trailing drop Drop being interrogated Leading drop A a _Y Not sorted Purity mask Not soriga ae Purity mask If the purity mask were set to 8 for the same target particle the non target particle would fall
234. tee he ease ha E ne Ed 183 Purging the Fluid Filters 2 0 0 ccc cence ens 190 Purging the Sheath Filter 2 0 ccc ccc een en enns 191 Changing the Fluid Filters 2 0 ccc cc cece nes 191 Changing the Sheath Filter 2 0 cc ene eens 192 Changing the Ethanol Shutdown Filter 0 0 00 e eee eee ee 193 Changing the Sample Lines 0 ec cece cee ee een nes 194 Changing the Air Filters 0 0 ccc cc een ene nnes 202 viii BD FACSAria II User s Guide Changing the Sheath Tank Air Filter 2 0 0 cece ec cee eee 203 Checking the Fluidics Cart Drip Tray 2 0 cece cece eee eens 203 Unscheduled Maintenance 0 ccc cece eee nen ene nees 204 Changing the Integrated Nozzle occ ce eens 205 Cleaning the Integrated Nozzle 2 00 ccc ce eens 206 Using the Standard Nozzle 6 cence eens 208 Temporary Replacement of a Seal 0 cece ccc ee ee ene 208 Installing an O ring in a Standard Nozzle 2 0 0 0 ec eee eee es 209 Closed Loop Nozzle Maintenance 00 ce cece eee eee eee nee 210 Installing or Removing a Sample Line Filter 02005 212 Changing the Pinch Valve Tubing 0 0 0 ccc eee cee eens 214 Cleaning the Camera Windows 0 00 cece cence nent e nee 217 Removing the Deflection Plates 0 ccc cece eee n nee 219 Lubricating the Sample Injection Chamber O Ring 0000 220 Using Custom Optical Filters 2 0 cee eee 222 Cleaning the Op
235. ter is set to a tube Stop Acquiring Stops sample acquisition without unloading the sample tube The Acquire Data button functions only when a tube is loaded Flow Rate Controls sample flow rate from 1 0 11 0 10 80 pL min 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 II User s Guide Sorting Controls All sorting on the BD FACSAria II cytometer is controlled by BD FACSDiva software Sorting controls are shown in Figure 3 8 on page 83 See Table 3 1 on page 83 for explanations of each area Figure 3 8 BD FACSDiva sorting controls 6 0 Table 3 1 Description of sorting controls O The Sort menu provides access to the 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 84 The Sort Layout window designates which device will be used to collect sorted particles and which particles will be sorted into each sort location Sorting controls and counters are also displayed in the window See Sort Layout on page 87 Chapter 3 Using BD FACSDiva Software 83 Table 3 1 Description of sorting cont
236. ter quality control QC to ensure consistent performance over time During cytometer QC you want to keep as many variables as constant as possible For example always use the same QC particle type and lot number sheath pressure and PMT settings If you work with different sheath pressures you might consider having a separate QC experiment for each pressure When the experiment cytometer settings and QC sample are constant changes in the recorded means and coefficients of variation CVs indicate changes in cytometer performance over time QC data should be analyzed for trends over 30 60 runs This section describes how to use a QC experiment template to verify 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 for cytometer QC see Cytometer Setup Particles on page 261 NOTE QC results are affected by laser and fluidics performance We strongly recommend following the laser and fluidics maintenance procedures in Shutdown and Maintenance on page 175 Setting Up the Cytometer Configuration The cytometer configuration includes these settings e Fluorochromes e Filters and mirrors e Sheath pressure must match sheath pressure in sort setup e Nozzle size e Window extension 308 BD FACSAria II User s Guide The steps in this section show you how to set up the cytometer configuration in preparation for performing QC 1 Sele
237. ternal 4 filters changing 202 line 102 180 pressure 5 supply external 4 AMO See aerosol management option amplitude about 47 adjusting 48 105 14 206 325 analysis batch 146 data 138 printing 144 sorting 143 151 161 application settings about 123 adjusting area scaling 124 creating 123 optimizing PMT voltages 128 saving 130 window 133 applications custom 74 recommended flow rates 31 recommended sort setup 85 area parameters 42 area scaling adjusting 310 primary laser 312 arrays detector 22 34 252 aseptic sorting 187 aspirator drawer about 15 opening 50 92 164 assistance technical xv attenuation control 50 auto drop delay about 52 graph 160 overview 159 using 159 autoclaving sheath tank 187 automated cell deposition unit ACDU about 17 accessing stage 92 chamber 17 collection devices 171 custom devices 172 installing splash shield 168 326 BD FACSAria II User s Guide sorting 168 troubleshooting 233 backflush sample line 184 bandpass filters 37 base configurations 76 batch analysis performing 146 BD FACSAria II about 1 cytometer 6 fluidics cart workstation 26 BD FACSDiva software See software beads Accudrop 156 calibration 261 CS amp T 120 setup 118 beam splitters 36 biexponential display 141 sort gates 161 bleach fluid line 189 bleeding filters 190 breaker circuit 24 breakoff drop 45 setting up 105 107 troubleshooting 105 231 window 46 84 bulk sorting 152 b
238. the screws when the stream is centered in the aspirator For further assistance see Troubleshooting the Stream on page 226 Chapter 6 Shutdown and Maintenance 207 208 Using the Standard Nozzle The standard nozzle has a replaceable O ring in contrast to the integrated nozzle which has a seal fixed in place This section contains information on handling the standard nozzles particularly in dealing with the standard O ring Changing the Standard Nozzle The procedure to change the standard nozzle is the same as that for the integrated nozzle except to verify that the O ring stays in place See Changing the Integrated Nozzle on page 205 While removing the nozzle in step 2 observe the following process 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 To locate and remove the O ring wet the wooden end of a cotton swab and use it to wipe the inside of the nozzle well Use the magnifying glass in the accessory kit if necessary Cleaning the Standard Nozzle The procedure to clean the standard nozzle is the same as that for the integrated nozzle except that you remove the O ring before sonicating and replace the O ring afterwards See Cleaning the Integrated Nozzle on page 206 The same practice applies for the closed loop nozzle See Cleaning the Integrated Closed Loop Nozzle on page 210 Te
239. the splash shield Ensure that an air filter is installed in the sort collection chamber door The filter traps airborne dust that could clog the ULPA filter Note that the filter should be changed on a monthly basis See Replacing the Air Filter on page 288 Air filter Close the sort collection chamber door NOTE The sort collection chamber door must be closed for the evacuator to generate negative pressure in the chamber Appendix C BD Aerosol Management Option 279 4 Switch on the main power on the back of the evacuator Figure C 4 on page 280 Figure C 4 Turning on main power Main power Power button 6 Press the up or down arrow button to set the suction control rate to 20 Each time either arrow button is pressed the suction will increase or decrease by 10 When two lights are lit on the suction control indicator the actual air flow is the value between the two illuminated percentages A Do not set the suction control rate above 20 Higher rates could affect the stability of the side streams 7 Verify that the filter flow gauge reads less than 2 4 inches of H O Figure C 5 on page 281 For a new filter the gauge should read 1 1 1 4 inches of HO As the filter is used the reading will increase If the gauge reads 2 4 inches of HO or greater replace the filter See Replacing the ULPA Filter on page 284 280 BD FACSAria II User s Guide Figure C 5 Reading the filter flow gauge
240. tical Filters 2 0 0 0 cece eee ee teen eee 223 Removing or Installing the FSC ND Filter 0 0 0 ccc eee eee 223 Chapter 7 Troubleshooting 225 Troubleshooting the Stream 0 0 ccc cee nen eens 226 Troubleshooting the Breakoff 0 ccc ccc cece nee nee 231 Sorting Troubleshooting 0 0 ccc cee tence een eens 232 Acquisition Troubleshooting 00 cece eee eee eee eens 237 Fluidics Troubleshooting 0 0 c cece cee cence tent en tenes 244 Electronics Troubleshooting 0 000 cece cece tenes 245 Chapter 8 Technical Specifications 247 Cytometer Specifications 1 6 0 cece cece ene n ene e eens 248 Environment 28 20 ig Ss te ees ade ae a a a Als 249 Performance f 25yteaw a6 eden tena dss ae E lane ae Glebe 249 Sort Performances sis athe a Bat hn Rated Cae teeta oor 250 Contents ix Excitation Optics 4 6 S43 4 3 4sne Sob saws SHES Saad SSS Soa EAN 251 Emiussion Optics oeae eu es p AE tesWwew ee ete oie E iia 252 Fluidics Cart Specifications 2 1 cece ccc een ence e eens 254 Appendix A Supplies and Consumables 255 Cytometer Supplies s riei ea cece eee eee ence e eens 256 Optical Components sinus ces ye eee PA eee Ee 6 OLN we 256 Accessory Kit cea tee kk beri dare ae hand died a WA EH a Sa ee eh ae ee GA 258 Other Replacement Parts 0 cece cece cece ence cence nees 260 Consumables edun estar tes os wie ates es Ca ee SR SA ee eS 261 Cytometer Setup Particles 0 ce
241. ting infection Dispose of waste using proper precautions and in accordance with local regulations Wear suitable protective clothing eyewear and gloves Change the air filter in the sort collection chamber door on a monthly basis The filter traps airborne dust that could clog the ULPA filter Regular replacement of the air filter will extend the life of your ULPA filter See Replacing the Air Filter on page 288 Do not touch the Filter Life Reset button during normal operation Doing so could shut down the evacuator and prevent the collection of aerosols Appendix C BD Aerosol Management Option 283 284 Do not disconnect the tubing from the instrument manifold outlet or the ULPA filter unless you are changing the filter Repeated removal and reattachment of the tubing could loosen the connection and disrupt airflow e To ensure optimal airflow keep the tubing free of kinks and away from sharp or heavy objects Do not crush or puncture the tubing Ensure that the tubing is securely attached at both ends before turning on the evacuator power e Keep the sort collection chamber free of potentially obstructive debris such as Kimwipes or disposable pipettes Replacing the ULPA Filter Replace the filter when either of the following conditions occur e The filter flow gauge reads 2 4 inches of H20 or greater at 20 suction e The red filter life indicator LED is blinking NOTE When the red light turns on and is not blinking yo
242. tion 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 The home and farthest locations for a 96 well plate are A1 and H12 respectively 172 BD FACSAria II User s Guide Figure 5 8 Home and farthest location on 9 drop slide example Farthest Y O O O i Home 1 Select Sort gt Custom Devices 2 Click the Add button in the Custom Devices dialog 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 numbered 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 j Set Home Go Farthest j Set Farthest v Waa 3 Select the text in the Name field and enter a new name 4 Enter the number of sort location rows and columns A device can have up to 60 rows and 25 columns 5 Use the arrow buttons and the Test Sort button to set the home location then click Set Home Chapter 5 Sorting 173 See Setting Up the Stream on page 170 for details There are no default values for custom devices so greater initial adjustment with the arrow buttons is required 6 Use the same procedure to set the farthest location then click Set Farthest The farthest
243. tion and need only a quick reminder of the main steps use the quick reference guide provided with this user s guide xiii Conventions xiv The following tables list conventions used throughout this guide Table 1 lists the symbols that are used in this guide or on safety labels to alert you to a potential hazard Text and keyboard conventions are shown in Table 2 Table 1 Hazard symbols Symbol Meaning Caution hazard or unsafe practice that could result in material damage data A loss minor or severe injury or death A Electrical danger A Laser radiation Biological risk a Although these symbols appear in color on the instrument they are in black and white throughout this user s guide their meaning remains unchanged Table 2 Text and keyboard conventions Convention Use M Tip Highlights features or hints that can save time and prevent difficulties NOTE Describes important features or instructions 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 select File gt Print means to select Print from the File menu Ctrl X When used with key names a plus sign means to press two keys simultaneously For example Ctrl P means to hold down the Control key while pressing the letter p BD FACSAria II User s Guide Technical Assistance For technical questions or as
244. tion in order to record only singlet events This template contains cytometer settings and gated plots for this purpose A procedure is given in Recording Data on page 142 For experiments where you do not need to distinguish singlets from doublets you can start with a default experiment or a template of your choice To use the template select Experiment gt New Experiment and select the Doublet Discrimination Gating template Click OK to add a copy of the template to the Browser 132 BD FACSAria II User s Guide Experiment Templates General G5 Practice cad Name Date Name Doublet Discrimination Gating Blank Experiment Accudrop Drop Delay 11 22 06 11 00 AM Blank Experiment with Sample Tube 11 22 06 11 00 AM Doublet Discrimination Gating 11 22 06 11 00 AM 11 22 06 11 00 AM Name Doublet Discrimination Gating copies 1 IE Rename the experiment appropriately for example 4 Color experiment Right click the experiment level Cytometer Settings in the Browser and select Apply Application Settings See Figure 4 21 Figure 4 21 Application Settings window Application Settings Cytometer Configuration 7 i J v Name Owner Date Created 5 color Experiment_PM Administrator 04 05 07 12 18 31 PM See Application Settings on page 123 for instructions on creating application settings Select the application setting for your sample and click Apply See Figure 4 22 o
245. tive clothing and gloves Changing the Primary Sample Line To replace the primary sample line you will need a 12 inch length of replacement tubing from the accessory kit There is a different fitting at each end of the tubing so the procedure is divided into two sections Assembling the Collet Fitting at the Pinch Valve At the pinch valve end of the primary sample line a collet fitting joins the sample line to the pinch tubing To replace the tubing 1 Turn the stream off if needed Make sure the loading port is in the unload position 2 Unscrew the nut from the collet fitting and pull the nut and collet apart 3 Pull the pinch tubing out of the nut and then pull the sample line out of the pinch tubing See Figure 6 13 Chapter 6 Shutdown and Maintenance 195 4 Locate a new 12 inch piece of sample line tubing a Slide the nut over the new sample line tubing Figure 6 13 Collet nut fitting on pinch valve tubing Collet Nut Pinch valve tubing Sample line tubing LZ WZ 77 6 mm b Slide the pinch valve tubing over one end of the sample line tubing until approximately 6 mm of the sample line is inside the pinch valve tubing c Slide the pinch valve tubing on the inside of the teeth of the nut until it stops d Couple both pieces of the fitting together and then tighten until finger tight 196 BD FACSAria II User s Guide Prima
246. to the loading port Chapter 6 Shutdown and Maintenance 213 8 Click Done and the sample injection chamber raises A Be careful of a pinching hazard as the sample injection chamber is raised The sample line should not bow or bend when a tube is loaded 9 Adjust the sample line height if needed 10 Tighten the sample line fitting and click Done The bulk injection chamber is lowered and the sample purge mode is turned on for about 5 seconds until several drips exit the filter A message is displayed at the bottom of the wizard See Figure 6 27 on page 214 Figure 6 27 Sample line wizard message 63 Change sample filter complete Lowering the Bulk Injection Chamber Done Sample Purge ON mode in progress Done Waiting 5 sec to drip Done Sample Purge OFF mode in progress Done 11 Click Done and the process is complete Changing the Pinch Valve Tubing The tubing that runs through the pinch valve should be changed as needed The system ships with replacement tubing cut into 3 inch lengths found in the accessory kit 214 BD FACSAria II User s Guide Figure 6 28 Pinch valve tubing Pinch valve collet fittings Pinch valve tubing Pinch valve To replace the tubing 1 Turn off the stream if needed Make sure the loading port is in the unload position Pull the existing pinch valve tubing out of the slot in the pinch valve Grasp the tubing with two fingers on each side of the pinch valve and pu
247. tream is stable and the sort is not pausing and re run the auto drop delay 160 BD FACSAria II User s Guide Figure 5 5 Auto drop delay dialog Auto Drop Delay 18 40 94 39 18 37 97 91 18 34 94 73 18 31 94 86 18 28 83 29 18 67 Found tess than 90 0 exiting Delay 18 67 Drop detay is set to 18 4 Click Exit to exit or run again Sorting Before beginning the sort do the following 1 2 Perform the steps outlined in Setting Up for Sorting on page 150 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 143 and in Getting Started with BD FACSDiva Software NOTE Gates drawn on a biexponential scale can be used for sorting However the cytometer will sort on a log scale Therefore a gate that crosses the zero boundaries will sort all events below zero into that gated population This can cause a variance between the sort results and the statistical results in the software If the gate is completely below zero on a biexponential plot no events will be sorted NOTE Snap To gates cannot be used for sort gates Chapter 5 Sorting 161 Setting Up the Experiment Mi Tip 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 use the
248. tt r 3 0 3 2 08 ed e tas Ma eet Seo wh aes he a E CP AOS eae 32 Fluorescent Signals ss sraa aaan EEES UDELAT EENIA AUAA 33 Signal Detection se 3 44 53 Soh eda ee Seb Pe eeed COVE a ae Pea oe Se 34 Detector ATrAYS aeiee ot Sites tos Oe ase ete ee Loe oe ea dager 34 Filters 2 cde dtant hed btans oniatana eee tawas aba aba Sb 35 Detectors ria e Beeb ee ae eee a oh ea Bore heey Bae 40 Electronic Processing 3 22 s cc4 52205 sed sc Beda e Seed peaa k ESE GEESE 41 Pulse Parametets Saar sce edna e outa Saw wr beieigd bu E Sie Seam e as 42 kaser Delay sss0s Si 2 a a ees seb PAS ae TSE OR hos g Paves aa St 43 SORUNS fos atoe Sa ata ee weet ee Rena yee eh de hte ttc 44 Drop RormatGns i820 Sistasvece lan ee Bate os he Sa eA AS a E cave weet Ses 45 Side Stream Formation 0c cece eee een nent tence a a 49 Drop Charging serang cak eeii waa saclay cele eee Gare eRe 52 Conflict Resolution During Sorting 0 00 00 c cece ene 53 Chapter 3 Using BD FACSDiva Software 61 Workspace Components 0 c cece cece cece n eee n ene nees 62 Cytometer Controls esris a esGe bei et SE eee eS WE ed SS 63 Fluidics Controlsry eren en Phe ee ea ae ea ale Laie ata aes 63 Fluidics Level Indicators 0 ccc ee ccc eee ene e eens 67 Cytometer Configuration 2 0 0 0 ccc cee een tenes 68 Cytometer Status Report serrer sesirih Oriri eee tne tne ee eens 72 Custom Configurations 0 ccc cece cent eens 74 Acquisition Controls 2 2 0 cec
249. ttered and fluorescent light is collected by the fluorescence 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 The following sections describe 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 II flow cytometer octagon and trigon The octagon detects SSC and up to seven fluorescence signals excited by the 488 nm blue laser The trigons detect fluorescence signals excited by the 633 nm red and 405 nm violet lasers respectively Each trigon can detect up to three fluorescence channels An optical upgrade is required to fill all detection channels Figure 2 6 Octagon and trigon detector arrays EZ 919 Se hk 4 y Violet laser signal Red laser signal Blue laser signal Each detector array houses dichroic and bandpass filters which steer and filter the emitted light and photomultiplier tubes PMTs which detect light signals BD FACSAria II User s Guide Filters Optical filters modify the spectral distribution of light scatter and fluorescence directed to the detectors Three 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 and ne
250. typographical conventions xiv U Unload button 82 unloading tubes 10 30 user preferences 143 V View Configurations selection 68 viewing global worksheets 140 views See also windows sort layout 87 statistics 144 violet laser configuration 19 detector 22 34 257 271 laser delay 43 switching from near UV laser 270 voltage adjusting PMT 42 controls 50 optimizing PMT 128 warning light 15 Index 337 W waste aspirator 15 cap changing 115 container parts 114 emptying 114 water bath 297 303 width parameters 42 Window Extension adjusting 318 windows See also views about 62 83 Acquisition Dashboard 81 Breakoff 46 camera cleaning 217 CS amp T 117 cytometer configuration 69 device setup 171 showing 311 Side Stream 49 strobe lens 218 worksheets See also global worksheets printing 144 viewing 140 workspace components 62 83 setting up 131 setting up CSXT 117 workstation about 26 shutting down 177 181 Y Yield mask 54 56 mode 58 338 BD FACSAria II User s Guide
251. u have approximately 1 hour of filter life remaining If the red light comes on during sorting the filter will not stop working Replace the filter as soon as possible when the red light starts blinking To replace the ULPA filter 1 Turn off the evacuator main power and disconnect the electrical plug A To prevent potential shock always turn off the evacuator main power and disconnect the electrical plug from the power source before installing or removing any filter 2 Disconnect the tubing from the manifold Figure C 6 on page 285 The manifold is located in the connection panel on the left side of the instrument BD FACSAria II User s Guide Figure C 6 Disconnecting tubing side of instrument Remove the spring loaded filter hold down While pushing down on the filter pull up on the spring loaded handle Figure C 7A and guide the handle over the top of the filter and behind the metal plate in the back of the evacuator Figure C 7B Figure C 7 Removing the filter hold down Appendix C BD Aerosol Management Option 285 4 Liftoff the ULPA filter and attached tubing from the evacuator and dispose of both the filter and the tubing A All biological specimens and materials coming into contact with them can transmit potentially fatal disease Handle the ULPA filter attached tubing and all instrument hardware as if capable of transmitting infection Dispose of waste using proper precautions and in accordance with lo
252. ues are correct as part of your QC If adjustments are needed they should be slight The delay is properly adjusted when the fluorescence signal intensity is at its highest a Click the Laser tab in the Cytometer window b Change the window extension to 0 e Ifthe APC A signal stayed the same then go to step e e Ifthe APC A signal decreased then follow step c through step e c Click in the Delay field for the red laser Cytometer FACSAria 1 Cytometer Connected 318 BD FACSAria II User s Guide achieved Before adjustment d Adjust the laser delay value until the maximum APC A signal is After adjustment Rainbow beads High Sort Setup Rainbow beads High Sort Setup potatoe Count 10 L PE 34 Count PRP PP HEN EA FEA FA HEN AEA HA 50 100 150 200 250 APC A 1 000 mrri hr s0 100 150 200 250 APC A 1 000 Tube Name High Sort Setup Record Date Mar 28 2006 12 57 26 PM Tube Name High Sort Setup Record Date Mar 28 2006 12 57 26 PM SOP Administrator OP Administrator APC A APC H APC A APC H Population Mean Mean Population Mean Mean Wri 101 398 127 188 Mri 124 572 127 868 e Reset the window extension to the appropriate setting typically 2 Adjust the red laser area scaling until the APC A signal matches the APC H signal if needed Click the Laser tab in the Cytometer window e Increase the area scaling factor i
253. ut 6 Fill the water bath with distilled water containing 0 1 g L sodium carbonate Sodium carbonate helps reduce corrosion See the water bath manufacturer s documentation for fill levels and other setup information NOTE BD Biosciences does not recommend using ethylene glycol antifreeze in the water bath 298 BD FACSAria II User s Guide 7 Plug in the water bath power cord NOTE Do not start up the water bath until after you have connected the recirculating water tubing as described in the following sections Setting Up the Tube Holder This section describes how to install the temperature control tube holder on the instrument and how to attach the recirculating water tubing to the tube holder A 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 Place collection tubes in the temperature control tube holder Tube holders are available for 15 mL two way sorting 12 x 75 mm four way sorting and 1 5 mL Eppendorf tube four way sorting For compatible tubes see Supplies and Consumables on page 255 2 Attach the recirculating water tubing to the tube holder a 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 Note that the tubing ports are labe
254. utral density ND filters allow fine tuning of the spectral wavelengths that need to be collected Figure 2 7 Figure 2 7 Detectors and filters in an octagon array D Chapter 2 Theory of Operation 35 36 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 Figure 2 8 Light reflection by longpass filter Longpass 480 520 100 4 460 500 540 a eo Transmission o i T T 450 500 550 Wavelength nm 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
255. uttons See also controls Access Stage 92 chamber light 9 emergency stop 25 Load 82 power 24 Sort 92 Unload 82 waste drawer 50 C calculating compensation 135 cameras about 23 51 cleaning windows 217 cap waste 115 cart fluidics See fluidics cart chambers ACDU 17 sample injection 9 sort block 13 sort collection 17 changing air filters 202 203 ethanol shutdown filter 193 fluid filters 191 nozzles 205 optical filters 222 pinch valve tubing 214 sample lines 194 sheath filters 192 waste cap 115 charging drops 52 circuit breaker 24 cleaning camera windows 217 cytometer 181 183 flow cell 176 fluidics 187 mode failure 244 modes 183 nozzles 206 optical filters 223 strobe lens 217 clogged nozzle 206 closed loop nozzle cleaning 210 for shutdown 176 installing 178 179 maintenance 210 replacing tubing 211 coefficient of variation CV collection devices 17 87 optics 20 tubes replacing 166 collet fitting 198 compensation calculating 135 controls creating 134 gating data 137 setup 137 components BD FACSDiva workspace cytometer 6 electronics 24 fluidics 7 optics 18 workstation 26 computer about 26 shutting down 177 181 configuration cytometer about 40 68 69 adding 74 and sort setup 79 copying 76 custom 74 editing 77 laser 266 mismatch dialog 80 selecting 118 conflicts sort about 53 counting 93 high 241 62 83 Index 327 printing 94 saving 91 162
256. w the current sheath pressure setting using the Cytometer gt Sheath Pressure command Figure 2 2 Figure 2 2 Sheath pressure level F Sheath Pressure Sheath Pressure Adjustment Sheath Pressure 70 00 Sit After fluidics startup sheath flow is controlled using the Stream button in the Breakoff window Figure 2 3 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 dialog See Drop Formation on page 45 for a complete description of the Breakoff window Figure 2 3 Stream control in Breakoff window Pa A Stream gt J Sweet Spot As a general rule the sheath pressure level is set by selecting a sort setup mode from the Sort menu rather than by adjusting the Sheath Pressure control Each sort setup mode is optimized at a preset sheath pressure If you change the sheath pressure a multitude of other values will be affected and need updating including the drop drive frequency drop charge levels laser delay area scaling factor and other values For more information see Sort Setup on page 85 Chapter 2 Theory of Operation 29 30 Sample Flow Sample is introduced into the cuvette when the Load button is clicked in the Acquisition Dashboard Figure 2 4 After Load is clicked the loading port rises to enclose the tube within the sample injection chamber The chamber is automatically pressurized
257. xported comma separated value CSV files can be opened with a spreadsheet application such as Microsoft Excel BD FACSAria II User s Guide Templates When you install BD FACSDiva software for the BD FACSAria II cytometer the following additional experiment templates are installed in the BD Export Templates directory Accudrop Drop Delay template Contains a single specimen and tube a gated plot on a standard worksheet and a predefined sort layout This experiment is used for setting the drop delay during sorting as described in Determining the Drop Delay Manual Method on page 154 Doublet Discrimination Gating template Contains custom cytometer settings a single specimen and tube and three gated plots on a global worksheet This experiment is used to eliminate doublets as described in Data Collection on page 131 QC Experiment template Contains cytometer settings a single specimen one tube and two global worksheets containing gated plots and statistics for data from each laser This experiment is used for daily cytometer quality control as described in QC Using BD FACSDiva Software on page 307 Experiment Templates pee General G5 Practice a Name Name Accudrop Drop Delay Blank Experiment f4ccudrop Drop Delay 6 11 00 4M Blank Experiment with Sample Tube 11 22 06 11 00 AM Doublet Discrimination Gating 11 22 06 11 00 AM QC Experiment 11 22 06 11 00 AM Name Accudrop Drop Delay Chapter
258. zing PMT Voltages 1 Right click Cytometer Settings in the Browser then select Application Settings gt Create Worksheet A second global sheet is added with the plots created according to your selections in the parameters tab See Figure 4 19 Use the gray boxes and crosshairs to guide your optimization Figure 4 19 Optimizing PMT voltages All Events All Events All Events lt a a 2 f 7 a a a Ta Ta a an 50 100 150 200 250 p0 10 0 t p0 10 0 t FSC A 1 000 FITC A APC A 2 Load the unstained control tube onto the cytometer 128 BD FACSAria II User s Guide In the Cytometer window optimize the settings for your application a Optimize the FSC and SSC voltages to place the population of interest on scale b Optimize the FSC threshold value to eliminate debris without interfering with the population of interest c If needed increase the fluorescence PMT voltages to place the negative population within the gray boxes Try to align the center of the negative population with the crosshair shown in the gray box NOTE Do not decrease the fluorescence PMT voltages at this point Doing so may decrease your ability to resolve dim populations from the negative population Unload the unstained control tube from the cytometer Load the multicolor sample onto the cytometer Verify that the positive populations are on scale If a positive population is off scale lower the PMT vo

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