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BD FACSCanto flow cytometer reference manual

1. I Show Grid Background Color Population Draw a All Events M Be 1 Gridlines are used to delineate log decades on plots In a four log display values are displayed from 26 262 143 Thus the first log decade ranges from 26 262 Thelines are shown only in plots that display log parameters O ptimize the voltages to place the negative population for each fluorescent parameter within the first log decade Figure 3 4 on page 78 Chapter 3 Running Samples 77 Figure 3 4 Unstained Control Tube after PMT adjustment Unstained Control Unstained Control 250 SSC Ax 1 000 Count 200 250 x 1 000 FITC A Unstained Control Unstained Control Count 107 10 10 PerCP Cy5 5 A Unstained Control Unstained Control Count 10 104 PE Cy A Unstained Control 107 10 10 APC Cy A 78 BD FACSCanto Flow Cytometer Reference M anual 10 Click Record 11 After all events have been recorded click Remove Tube A It is critical that you follow the tube removal sequence exactly Failure to follow this sequence could result in carryover between samples A progress dialog appears Progress YD Remove tube in progress Do notload next tube until flush is complete e Hold your sample tube with one hand while you push the aspirator arm all the way to the left with the other hand Always hold onto your sample tube when you push the aspirator arm to the side If you move the arm
2. GAD Dd 204 BD FACSCanto Flow Cytometer Reference M anual Figure F 3 Example of QC log in progress H a L semu sojered E MLI ET Er oe Teee E Terea sase anjg ft ec e E ee he2 1777 8 papapapa a ass mez edil aa pee rafie etree ie Appendix F QC Log 205 206 BD FACSCanto Flow Cytometer Reference M anual Index A aborts electronic troubleshooting 146 accessory kit 176 acquisition controls 52 events to record 84 troubleshooting 143 Acquisition Templates creating 83 previewing data 83 90 adapter lever 28 adjusting flow rate 53 PMT voltages 75 threshold 75 air filter 108 airlock removing 128 alcohol isopropyl 118 analysis data 83 immunophenotyping 87 reusing 90 saving 90 Analysis objects copying 90 area scaling troubleshooting 147 arrays detector 32 aspirator arm 28 assistance technical xiv automatic clean 44 auxiliary air supply switch 38 B Bal seal 120 bandpass BP filters 157 beads running setup 62 setup 61 bleeding filters 105 breaker main circuit 35 bubble filter 117 Bubble Filter Purge 117 button power 35 5 7 bypass tubing installing 128 129 C cables reconnecting 125 calculating compensation 80 81 cap waste tank 104 changing cubitainers 98 circuit breaker cytometer 35 fluidics cart 38 fluidics cart resetting 124 Clean Flow Cel 116 207 cleaning automatic 44 decontaminate fluidics 107 external surfaces 118
3. Install saved dat file or contact BD Biosciences H igher than expected CVs for some brightest populations during DN A analysis Possible preamp non linearity with very bright signals Set G G diploid population peak at channel 25 000 or less Chapter 5 Troubleshooting 141 Fluidics Cart Troubleshooting Observation Water leakage around fluidics cart base Possible Causes e Normal condensation overflow from pressure relief valve e Extremely humid climate Recommended Solutions 1 Turn off the cytometer power 2 Clean up the water 3 Turn off the power to the system daily This empties the condensation trap in the fluidics cart Bleeder valve on fluidics cart filter open 1 Turn off the cytometer power 2 Clean up the liquid 3 Check and close all bleeder valves See Purging the Fluidics Filters on page 105 Broken fluid line Contact BD Biosciences Fluidics cart will not power on cytometer on Circuit breaker switch to cart off Toggle the cart circuit breaker on Auxiliary air supply switch on cart not normally connected to auxiliary air Toggle the auxiliary air supply switch off When the auxiliary air supply is switched on the cart s Own air pumps turn off Power cord from fluidics cart to cytometer disconnected M ake sure both ends of the power cord are connected Fuse blown Replace the fluidics cart fuses See Replacing the Flu
4. aone GLa ia 503 value of Y 3 4 2 Standard error of estimate of the data 08 2 22 07 97 1 91 Correlation 0 986 0 995 0 993 0 968 coefficient 186 BD FACSCanto Flow Cytometer Reference M anual Notes e Thecomparative method used in regressions was BD FACSCalibur with BD Simulset software and BD Simultest IM K Lymphocyte kit e Themethod used to fit the linear regression for all subsets was least squares A log of the least squares was used for CD4 and CD19 to stabilize the variance e Accuracy studies were conducted over a period of 35 days at three sites e Thenumber of replicate determinations used to calculate each mean for X Four replicates for CD3 one for all other subsets e Thenumber of replicate determinations used to calculate each mean for Y One to two replicates for all subsets e A difference exists between measurements of natural killer N K cell CD16 56 populations on the BD FACSCalibur with BD Simulset software and the BD FACSCanto with BD FACSDiva software BD FA CSDiva software uses a polygonal gate that allows more flexibility to accommodate the population shape This gate provides a tighter fit around the NK cluster and may exclude extraneous cells included with the rectangular gating provided by BD Simulset software Appendix E Performance Data 187 Individual lymphocyte Subset Accuracy Scatter Plots for Lyse Wash Method Subset Percentages CDa CD4 50 a0
5. software I BD FACSCanto clinical software B BD FACSDiva software 56 BD FACSCanto Flow Cytometer Reference M anual Instrument Startup Follow these steps to start up your BD FACSCanto flow cytometer l Turn on the main power The main power button is on the left side of the cytometer The button turns on power to both the cytometer and the fluidics cart main power button A To prevent fluid overflow make sure there is no tube on the SIT at startup 2 Start up the computer launch BD FACSCanto clinical software Double click the application shortcut on the desktop All BD Fa clsCanta Software Chapter 3 RunningSamples 57 3 AttheLog n dialog box choose your user name enter your password and click OK User ID WEE t Password For instructions on creating a user name and password refer to the BD FACSCanto Clinical Software U ser s G uide 4 Makesure the software is connected to the cytometer if necessary choose Cytometer gt Connect Remaining warm up time 04 30 p 00 03 O Connected status bar located at bottom of main window 5 Check fluid levels Low fluid levels or a full waste container are indicated by red Status Ox Parameter Value Loader Status Door Closed Vacuum Status Ok Pump Status Ok Float Status Ok FACSFlow Level 55 FACSFlow Pressure 4 5 Waste Tank Level 45 Shutdown Solution Level Empty Cleaning Solution Level Ok Laser Power Blue 0 Laser Current
6. 2 Create two Tubes under the LWB Specimen rename the Tubes appropriately For example TBNK_001 and TBNK_002 To create a second Tube select the Specimen and click T 3 Createa global worksheet rename it Record D ata e Ifthe Default global worksheet preference is enabled in User Preferences default option the global worksheet is already present Expand the Global Worksheets folder to locate and rename the worksheet e If the Default global worksheet preference is disabled create a worksheet by clicking the N ew Global Worksheet button in the Browser toolbar You can create up to ten global worksheets per Experiment 4 Usethe Experiment Layout dialog box to define labels and to specify the number of events to record for each Tube Parameter labels are defined in the Experiment Layout view Labels will appear on the plot axes and in all statistics views e Choose Experiment gt Experiment Layout e On theLabels tab enter appropriate labels for the Tube For example enter CD45 in the FITC field use the Tab key to move to the next field Experiment Layout Labels Keywords Acquisition Label ia 6 Color Expt oO OB U TBNK_004 F FITC PE PerCP Cy5 5 PE Cy7 APC APC Cy7 CD45 CD16 56 cbs cD19 CD3 cD4 FITC PE PerCP Cy5 5 PE Cy7 APC APC Cy7 CD45 CD16 56 CDS CD19 CD3 CcD4 F U TBNK_002 BD FACSCanto Flow Cytometer Reference M anual e OntheAcquisition tab enter 10 000
7. Figure 2 7 Acquisition controls unique to the BD FACSCanto FEE Acquisition Controls Tube_0O01 0 evtis 00 00 00 E Acquire E Record Storage Gate E All Events Stopping Gate E All Events x Events To Record 30000 evt Y Events To Display s500 evt Y Flow Rate Medium e Remove Tube starts countdown for manual cleaning of the SIT In combination with the aspirator arm movement the Remove Tube button initiates cleaning of the SIT to eliminate carryover between samples The button must be clicked each time you change a tube according to the following sequence A It is critical that you follow the tube removal sequence exactly Failure to follow this sequence could result in carryover between samples 52 BD FACSCanto Flow Cytometer Reference M anual Click Remove Tube A progress dialog appears Progress YD Remove tube in progress Do notload next tube until flush is complete Hold your sample tube with one hand while you push the aspirator arm all the way to the left with the other hand Always hold onto your sample tube when you push the aspirator arm to the side If you move the arm without holding the tube the tube could fall off the SIT and expose you to potentially blohazardous sample Remove the tube from the SIT Release the aspirator arm SIT cleaning occurs when the aspirator arm comes to center When the Progress dialog disappears you can load the next tube on
8. Fluidics cart side air vent 20 cm 7 9 in between air vent and other objects or wall to permit proper air flow Fluidics cart door air vent 20 cm 7 9 in between door and other objects or wall to permit proper air flow 45 4 kg lt 100 b fluidics cart only excluding tanks 81 7 kg lt 180 Ib with tanks full Receives power from cytometer Current 2A at 100 and 115 VAC 1A at 230VAC Consumption 175W No air or vacuum required Room air and vacuum can be attached 20 L Dil 5L 10 L BD FACSCanto Flow Cytometer Reference M anual Appendix E Performance Data This appendix covers the following topics e BD FACSCanto System Accuracy on page 186 e BD FACSCanto System Precision on page 195 e BD FACSCanto System Linearity on page 198 e BD FACSCanto System Carryover on page 200 185 BD FACSCanto System Accuracy Lyse Wash Method Subset Percentages Table E 1 BD FACSCanto system accuracy with BD FACSDiva software Measurement D3 CD4 cCD8 CD19 CD16 56 unit 0 99 1 03 0 98 1 01 0 96 Slope of fitted linear regression line Confidence 0 97 1 06 Interval Intercept of fitted linear regression line Confidence 0 31 4 11 1 03 0 02 0 83 0 44 1 28 0 41 Interval Total points 12 128 12 12 128 used in regression R ange of Data lowest highest 378 93 55 0 0 63 0 15 0 80 0 1 0 36 0 3 0 62 0 value of X R ange of Data OWE MONET lea 7 030 dese
9. Once the sample moves into the flow cell fluorescently tagged particles move in single file past the laser beam The emitted light from these particles provides information about their size shape granularity and fluorescence intensity The flow cell is beneath the flow cell access door Figure 1 3 Flow cell where lasers intercept sample stream obscuration bar FSC diode For more information see Fluidics System on page 150 Chapter 1 Introduction 29 Optics Excitation optics bring light to the flow cell Collection optics gather the light emitted or scattered by the tagged particles and convert them from optical to electronic signals The BD FACSCanto cytometer uses innovative designs for both the excitation optics and collection optics Some of the collection optics can be viewed by Opening the optics access door Excitation Optics The excitation optics consist of lasers fiber optic cables beam shaping prisms and an achromatic focusing lens as shown in Figure 1 4 on page 31 The BD FACSCanto instrument uses low powered air cooled and solid state lasers that do not have special power and cooling requirements acer Wavelength Min Power Commonly Used nm mW Fluorochromes Coherent Sapphire 488 blue 20 FITC PE PE Texas Red Solid State PerCP PerCP Cy5 5 PE Cy7 Pl JDS Uniphase HeNe 633 red 17 APC APC Cy7 Air Cooled a Measured out of fiber optic cable b Fora list of
10. FACSC anto a 40 FACSC anto BO Ce 20 40 o 40 so 80 o 20 40 60 FACSCalibur FACSCalibur 6 DS COW S0 40 50 si Ga FACSCanto e 38 40 20 10 20 o 20 40 60 a0 o 1 20 30 4 FACSCalibur i FACSCalibur t CD16 56 50 patient normal a 40 regression line FACSCanto confidence limit H ideal line 20 o 20 a Bc FACSCalibur 188 BD FACSCanto Flow Cytometer Reference M anual Lyse No Wash Method Absolute Counts Table E 2 BD FACSCanto system accuracy with BD FACSCanto software ni i mai Unit 0 99 0 97 0 96 1 02 0 95 Slope of fitted linear regression line Confidence 0 96 1 02 0 93 1 00 0 92 0 99 Interval Intercept of fitted linear regression line cells uL Confidence 53 20 5 51 Interval n n 40 70 27 00 i Range of data 105 0 4929 1 3 2 1 1909 1 13 3 lowest highest 3441 8 1459 6 value of X cells uL cells uL 2 6 points used in Total number of 108 regression Standard error of estimate of the data cells uL 128 4 97 8 38 3 33 4 Correlation 0 987 0 991 0 983 0 990 0 981 coefficient 8 0 we R ange of data TOA D 0 0 11 0 lowest highest 5257 3 3210 8 2526 7 1374 1 value of Y cells uL cells uL 1 4 Appendix E Performance Data 189 Notes e Thecomparative method used in regressions was BD FACSCalibur with BD M ultiset software and BD M ultitest IM K Kit with BD Trucount tubes e The least squa
11. cap or level sensor cap Chapter 4 Maintenance 103 3 Remove the disposable waste cap large sized cap and the attached trap from the container place the assembly on the bench label side up A Do not wet the cap If you see liquid inside the trap remove the drain plug and fully drain the liquid before you replace the plug 4 Empty the bleach exposed waste 5 Add approximately 1 L of bleach to the empty waste container 10 L container 6 If one month has passed since the last cap change detach it from the trap and replace it with a new cap A To prevent tank overpressurization 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 Vv Tip Write the date on the cap as a reminder space for date 104 BD FACSCanto Flow Cytometer Reference M anual 7 Screw the cap assembly onto the tank and hand tighten until it is fully closed 8 Attach the sensor and quick disconnect coupling to their ports Purging the Fluidics Filters Once a week purge air from all fluid filters by opening the bleeder valve on the top of each filter one at a time This ensures that the filters will not dry out Materials N o tools are required use your hands to open and tighten the bleeder valve e paper towels e proper protective equipment Procedure 1 Place a few paper towels beneath the filter Some fluid leakage might occ
12. directed to the detectors When photons encounter an optical filter they are either transmitted absorbed or reflected Figure A 6 Appendix A Technical Overview 155 Figure A 6 Effect of an optical filter on incident photons photons absorbed photons a transmitted reflecte a Ds Two kinds of filters are used on the BD FACSCanto flow cytometer default configuration longpass LP e bandpass BP On the BD FACSCanto flow cytometer the LP filters are called longpass dichroic mirrors Dichroic Mirrors 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 not all LP filters can be used as beam splitters 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 well controlled reflective characteristics Also optical filters and beam splitters are rated at a specific angle of incidence Their optical properties are therefore designed for that angle of incidence Longpass Filter In general LP filters pass wavelengths longer than the filter rating For example a 500 LP filter permits wavelengths longer than 500 nm to pass through it and 156 BD FACSCanto Flow Cytometer Reference M anual either absorbs or reflects wavelengths shorter than 500 nm Figure A 7 on page 157 Figure
13. these steps 1 Check the BD FACSFlow solution cubitainer connections to the fluidics cart M ake sure the tubing is attached See Changing a Cubitainer on page 98 2 Check the cytometer to fluidics cart connections See R econnecting the Fluidics Cart Tubing on page 125 3 Check all tubing for kinks BD FACSFlow cubitainer empty Replace the BD FACSFlow cubitainer See Changing a Cubitainer on page 98 Air in BD FAC SFlow filter on fluidics cart Purge air from the BD FACSFlow filter See Purging the Fluidics Filters on page 105 Air lock in BD FAC SFlow filter on fluidics cart See Removing an Air Lock on page 128 138 BD FACSCanto Flow Cytometer Reference M anual Instrument Troubleshooting continued Observation Fluid backfill into sample tube Possible Causes Cracked tube Recommended Solutions e Use anew tube for the sample e M ake sure you are using the correct tubes See System Requirements on page 40 Bal seal worn Install a new Bal seal See Replacing the Bal Seal on page 120 Air lock in BD FAC SFlow filter See Removing an Air Lock on page 128 Remove tube sequence not followed correctly cytometer still in acquisition mode e For BD FACSDiva software perform the tube removal sequence again step 10 on page 79 even if a tube is no longer on the SIT e For BD FACSCanto clinical software refer to the BD FACSCanto Clinical Software Use
14. voltage adjusting PMT 75 compensation and 79 fluidics cart fuses 136 W waste container described 37 emptying 102 waste tank cap 104 window extension troubleshooting 146 windows See also frames views workspace components 42 workstation shutting down 94 starting 57 Index 213 214 BD FACSCanto Flow Cytometer Reference M anual
15. Analysis object under global Record Data worksheet 3 On the Worksheet toolbar click the Global Worksheets button to switch to the Worksheet view 4 Create anew worksheet for the destination Tube rename the worksheet appropriately 90 BD FACSCanto Flow Cytometer Reference M anual 5 Select the Tube in the Browser right click the Tube icon and choose Paste The elements on the global worksheet are copied to the new worksheet You can view the analysis by double clicking the Tube in the Browser M Tip Apply the analysis to multiple T ubes by selecting more than one T ube Do not select any analysis objects along with the Tubes Enable the T ube specific worksheet user preference to automatically create a new worksheet for the pasted Analysis objects Automatically save a copy of the analysis with each Tube by enabling the Save Analysis After Recording preference before you record Tube data In this case the analysis plots are placed on the worksheet that is open at the time of recording To control where the plots are placed create a new worksheet before data is recorded Logging Out W hen you are finished using BD FACSDiva software but not ready to shut down the system choose File gt Log Out The BD FACSDiva workspace is hidden and the Log In dialog appears LOG IN Mame Maria Smith T Password This leaves the system available for the next operator to log in To shut down the system at the end
16. Blue 1 57 Laser Power Red 27 2 Event Rate 500 Sample Pressure 7 9 Tubes Since Last Clean 10 Cybtometer Setup Age hh mm 00 00 If necessary service the fluid containers as described in Changing a Cubitainer on page 98 or Emptying the Waste Container on page 102 58 BD FACSCanto Flow Cytometer Reference M anual 6 If automatic cleaning is disabled choose Cytometer gt Fluidics Startup If automatic cleaning is enabled fluidics startup runs automatically when the cytometer connects to the workstation Fluidics startup removes BD FACS shutdown solution from the fluid lines and replaces it with BD FACSFlow solution It takes about 4 minutes to complete 7 When Fluidics Startup completes click OK 8 Check the flow cell for air bubbles Lift the flow cell access door to see the flow cell check here for bubbles If you see bubbles remove them as described in Removing Bubbles from the Flow Cell on page 115 9 Check that the laser warmup has finished When laser warmup Is complete a Ready message appears You have finished starting up the cytometer Chapter 3 Running Samples 59 60 NOTICE You can also run startup using BD FACSDiIva software Startup instructions and commands are identical with the exception of the Cytometer menu which is called the Instrument menu in BD FACSDiva software Cytometer Instrument uidics Startup Standby Fluidics Shutdown Setup Cleaning Modes b Automatic
17. Bo a0 FACSCalibur CD4 30 60 FACS Canto J 4i 20 0 0 20 4i Bo a0 FACS Calibur CD19 40 FACS Canto a 20 FACSCalibur regression confidence ideal 194 BD FACSCanto Flow Cytometer Reference M anual BD FACSCanto System Precision Lyse Wash Method Subset Percentages Table E 4 Precision of the BD FACSCanto system with BD FACSDiva software Lymphocyte Within Run Within Run CV _ Total Precision Subset Precision SD SD cor foe pe pe p Total CV Notes e Testing was performed using commercially available normal and low CD4 control materials BD M ulti Check control and BD M ulti Check CD4 low control e The study was run over 20 days at one site and included 40 runs Three devices were used to collect data from control samples stained with two lots of reagent Appendix E Performance Data 195 Lyse No Wash Method Absolute Counts Table E 5 Precision of the BD FACSCanto system with BD FACSCanto software Lymphocyte N Winne RUN MC Total CV a LWitnin RuN MCL Total CV Subset cells uL cells uL cells uL cells uL e Testing was performed using commercially available normal and low CD4 control materials BD M ulti Check control M C and BD M ulti Check CD4 low control M CL e Thestudy was run over 20 days at one site and included 40 runs Three devices were used to collect data from control samples stained with one
18. Clean Fluidics Shutdown Instrument Configuration Cleaning Modes b Instrument Mame Instrument Status Report BD FACSCanto clinical software Instrument Setup p Standby BD FACSDiva software BD FACSCanto Flow Cytometer Reference M anual Instrument Quality Control Perform instrument quality control QC to ensure consistent instrument performance over time Performing Automated Setup Use the automated setup function within BD FACSCanto clinical software to adjust detector voltages to place channel specific setup beads at defined target values During the process spectral overlap values are also calculated and applied to compensate data for fluorescence spillover You must use BD FACS 7 color setup beads to perform setup Refer to the package Insert for preparation instructions Run setup once every 24 hours The software tracks the time between setups and displays it in the Status window A setup age of more than 24 hours appears in red Running a successful setup resets the timer Parameter Value Door Closed Vacuum Status Pump Status Float Status FACSFlow Level FACSFlow Pressure Waste Tank Level Shutdown Solution Level Cleaning Solution Level Laser Power Blue Laser Current Blue Laser Power Red Event Rate Sample Pressure Tubes Since Last Clean time since Cybtometer Setup Age hh mm 190 55 last setup Chapter 3 Running Samples 61 Running the Setup Beads Manual Mode 1 Prepare BD
19. FSC Area Scaling on page 169 Cannot delete from Parameters Threshold Compensation or Ratio tab views Row not selected Select the row using the selection button Recorded data in Tube M akea new Tube Chapter 5 Troubleshooting 147 148 BD FACSCanto Flow Cytometer Reference M anual Appendix A Technical Overview This appendix provides more information about these topics Flow Cytometry on page 150 Fluidics System on page 150 O ptics System on page 153 Electronics System on page 163 149 Flow Cytometry Flow cytometry measures certain properties of particles such as size and internal complexity using light To do this a flow cytometer needs a method to move the particles past a light source It also needs a way to collect and convert the light scattered or emitted by the particles into electrical signals M ost modern cytometers use lasers as the light source and transport the particles under investigation past the lasers using fluid or air The major systems in a cytometer that moves particles by fluid transport include e a fluidics system e an optical system e an electronics system Fluidics System A fluidics system in a flow cytometer moves particles in fluid through a flow cell past a laser beam and then into a waste tank Sheath Cubitainer to Flow Cell On the BD FACSCanto flow cytometer a separate fluidics cart houses the sheath cubitainer cleaning fluid cubitainers and t
20. a iow ween ae a ae ee aS 183 Fluidics Cart Specifications rerien albedo E tk Se oe Dee eae eS 184 CO CU eeta artes aar e ic ths apse ee decal oa AeA eet eth Poh shes 184 vill BD FACSCanto Flow Cytometer Reference M anual Appendix E Performance Data 185 BD FACSCanto System Accuracy 0c cee eee 186 Lyse W ash M ethod Subset Percentages 00 e eee ees 186 Lyse N o W ash M ethod Absolute Counts 00 eee eee 189 Lyse N o W ash M ethod Subset Percentages 00 cee evans 192 BD FACSCanto System Precision ce et tees 195 Lyse W ash M ethod Subset Percentages 0 0 cece eee ees 195 Lyse N o W ash M ethod Absolute Counts 0 0 eee 196 BD FACSCanto System Linearity 0 0 0 ce ees 198 BD FACSCanto System Carryover nussa aana aaea 200 Appendix F QC Log 201 Index 207 Contents IX X BD FACSCanto Flow Cytometer Reference M anual About This Guide This user s guide contains the instructions necessary to operate and maintain your BD FACSCanto flow cytometer M ost instrument functions are controlled by BD FACSDiva software and BD FACSCanto clinical software BD FA CSC anto clinical software contains modules for dedicated clinical applications with automatic gating algorithms while BD FACSDiva software Is non application specific You can use both softwares to perform instrument quality control Each software package has its own reference manual You ll find a desc
21. and workstations that 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 Biosciences is not liable for any claims related to or resulting from buyer user s failure to install and maintain virus protection History Revision Date Change Made 336928 Rev A 1 04 Initial release 337969 Rev A 4 04 Updated for CE IVD release 338619 Rev A 9 04 Updated for BD FACSDiva software 4 1 release Contents About This Guide xi CONVENON S ara eaa a E e N EAE es XII Technical ASSISTANCE esos kt ow ae PS ea hw Se a ee ee A XIV Safety and Limitations XV EE CAN SARE airna thet chat ca Gece desc cess eatae atari ten Utena te ates hea eis af oe XV BiOlOONCAI Safely minerer araa a wd aoa tSgededi adn se aoe dae ah ea eae a ek at XVI Laser Safa saciid Gs cat ae dee dowttants dekh ett i 8 ee oe Ave ad eed eet eas So ove ees XVII Laser PROC UCT Classificati h serinati GS Sans oh alain Sint Por ei oh cr atta Secchi XVIII Precautions for Safe O peration 0 cc ees XVIII Genel al S
22. flow cell 116 fluidics for storage 119 cleaning fluids not recommended 118 recommended 40 coefficient of variation CV high 146 compensation calculating 80 81 gating data 80 Setup 81 theory 161 163 Tubes creating 73 components BD FACSDiva workspace 42 FACSCanto system 26 fluidics cart 36 computer shutting down 94 starting 57 connecting power cords 35 controls acquisition 52 compensation 73 fluidics 43 Instrument 43 cord damaged xv creating Acquisition Templates 83 Analysis objects 87 compensation Tubes 73 cubitainer changing 98 described 37 cytometer components 27 not responding to software 139 power 35 setup 61 starting 57 D damaged cord xv data analyzing 83 87 gating 80 87 recording 83 85 Degas Flow Cell 115 detector arrays 32 dichroic mirrors 156 diode forward scatter 29 Diva software launching 57 login 58 logout 91 workspace described 42 208 BD FACSCanto Flow Cytometer Reference M anual doors access 27 flow cell access 27 how to open side door 109 optics 27 side 27 E electrical safety xv electronic aborts 146 electronics 163 ethanol 118 event rate 64 events not showingin plots 143 144 rate troubleshooting 145 troubleshooting 146 Experiment Layout 84 Experiments immunophenotyping 83 Sample optimization 70 F failed setup 66 fiber optics 31 filters air 108 bandpass 15 7 bubble 117 fluidic changing 111 fluidic purging 105 fluidic shown 36 l
23. fluid lines free of salt buildup do not exit the software or shut down the instrument until the fluidics shutdown procedure finishes Click OK when you see a message informing you that the system can be turned off Shutdown Status G tis now safe to turn of the system Chapter 3 Running Samples 93 10 Turn off the instrument main power A Turn off power to the cytometer at least once a day Doing so empties the condensation trap in the fluidics cart and prevents excess moisture from overflowing the trap or causing cart damage During shutdown you will normally hear a hiss caused by condensed water discharging from the fluidics cart pumps A If your laboratory runs the cytometer continuously and does not shut down at the end of the day toggle the Auxiliary Air Supply switch on for 15 seconds every 8 hours every 4 hours in an extremely humid climate Doing this empties the cart condensation trap and prevents excess moisture from overflowing the trap or causing cart damage 94 BD FACSCanto Flow Cytometer Reference M anual 4 Maintenance The BD FACSCanto requires little maintenance H owever to preserve the instrument s reliability you must regularly perform basic preventive maintenance This chapter explains the procedures you should follow to keep your instrument in good condition e Scheduled M aintenance on page 96 Unscheduled M aintenance on page 114 95 Scheduled Maintenance 96 For optim
24. gt Thearrow indicates a menu choice For example choose File gt Print means to choose Print from the File menu Ctrl X When used with key names a dash means to press two keys simultaneously For example Ctrl P means to hold down the Control key while pressing the letter p About This Guide xili Technical Assistance For technical questions or assistance in solving a problem e Read the section of the manual specific to the operation you are performing e See Chapter 5 Troubleshooting If additional assistance is required contact your local BD Biosciences technical support representative or supplier When contacting BD Biosciences have the following information available e product name part number and serial number e software version number e any error messages e details of recent system performance For instrument support from within the US call 877 232 8995 prompt 2 2 For support from within Canada call 888 259 0187 Customers outside the US and Canada contact your local BD representative or distributor xiv BD FACSCanto Flow Cytometer Reference M anual Safety and Limitations TheBD FACSCanto flow cytometer and the BD FACS Loader are equipped with safety features for your protection O perate them only as directed in the reference manual Do not perform instrument maintenance or service except as specifically stated If you operate this instrument in any way not specified in the
25. more information about label specific tubes 74 BD FACSCanto Flow Cytometer Reference M anual 3 Click OK when done A Compensation Specimen is added to the Experiment along with one Unstained Control Tube and a Stained Control Tube for each parameter that was specified in step 9 Worksheets containing the appropriate plots are added for each compensation Tube oe Compensation Specimen Instr Settings U Unstained Control U FITC Stained Control I PE Stained Control U PerlP Cy6 45 Stained Control I PE Cy Stained Control U APC Stained Control U APC Cy Stained Control He a E E Optimizing Instrument Settings W hen you performed instrument setup voltage settings were adjusted to set each parameter at a target value These settings might not be appropriate for the stained sample s you plan to analyze Before recording data you need to adjust FSC SSC and threshold settings gate on the population of interest such as lymphocytes and adjust voltages to optimize fluorescent signal For these adjustments you will need an unstained control sample It is important to perform these steps in order as some adjustments Influence others 1 Install the unstained control tube on the cytometer e Push the aspirator arm to the left e Place the tube onto the SIT and push up until the tube is firmly seated e Center the aspirator arm under the tube 2 Verify that the green Current Tube pointer is in front of the Unstained Cont
26. of the day see Daily Shutdown on page 92 Chapter 3 Running Samples 91 Daily Shutdown At the end of the day run the Fluidics Shutdown procedure to remove BD FACSFlow solution from all lines and rinse them with BD FACS shutdown solution This prevents saline crystals from clogging the fluidics lines and minimizes bacterial growth 1 2 a ua A W Install a tube with 3 mL of BD FACS cleaning solution on the SIT In the Acquisition Controls frame click Acquire HE Acquisition Controls Tube _001 0 evtis 00 00 00 Remove Tube W Record Storage Gate E All Events Stopping Gate E All Events Events To Record 30000 evt Y Events To Display 500 evt T Flow Rate Medium x After about 5 minutes click Acquire and remove the tube from the SIT Install a tube with 3 mL of DI water on the SIT Click Acquire After about 5 minutes click Acquire and remove the tube from the SIT To prevent fluid overflow do not leave a tube on the SIT during fluidics shutdown 92 BD FACSCanto Flow Cytometer Reference M anual Choose Instrument gt Fluidics Shutdown The following message appears Progress YD Fluidics is shutting down please wait Empty the waste and refill fluids if prompted to do so See Emptying the Waste Container on page 102 or Changing a Cubitainer on page 98 If you disconnect any fluid container for refilling prime the system before continuing To keep the
27. process control before you run samples 82 BD FACSCanto Flow Cytometer Reference M anual Data Recording and Analysis Once you optimize the instrument settings for your sample type you are ready to record and analyze data During analysis recorded data is displayed in plots and gates are used to define populations of interest You can use global worksheets to view and optimize data before it is recorded BD FACSDiva software analyzes the gated data and calculates statistics that you can print or export This section describes how to use BD FACSDiIva software features to record and analyze sample data As an example data will be recorded and analyzed for two Tubes of human peripheral blood stained with the following reagents e CD45 FITC e CD16 CD56 PE e CD8 PerCP Cy5 5 e CD19 PE Cy7 e CD3APC e CD4APC Cy7 Two strategies are shown for reusing Analysis objects If you are using a global worksheet to analyze data you can reuse the analysis strategy by displaying data for different Tubes on the same worksheet Alternatively you can copy Analysis objects to multiple Tubes at a time Setting Up the Global Worksheet This section shows you how to usea global worksheet to preview and record data for multiple samples To switch between the standard and global worksheet view click the Global Worksheets button on the Worksheet toolbar 1 Create a new Specimen rename the Specimen LWB Chapter 3 Running Samples 83 84
28. threshold gate population of interest adjust fluorescence PMT settings calculate compensation 170 BD FACSCanto Flow Cytometer Reference M anual Adding FSC A Before you begin an Experiment in which you will collect data based on FSC A you must add FSC A to the FSC parameter To do this follow these steps 1l In theInstrument frame click the Parameters tab Instrument FSC BSC FITC PE PearlP Cy5 4 PE Cy APC APC Cy7 eee LL 2 Select FSC A Instrument Status Parameters Threshold Compensation Ratio Laser Parameter Voltage Log AJ AW FSC A esc 240 aide 550 FITC 500 PE e PerOP Cy6 5 e PE Cy7 500 APC APC Cy7 500 HE Delete EEE Appendix B FSC Area Scaling 171 3 Do not deselect FSC H You will need FSC H selected to adjust FSC area scaling Adjusting the FSC Area Scaling Factor In the Acquisition Controls frame click Acquire to stop acquisition Create a dot plot on the Unstained Control worksheet M ake sure the plot axes are labeled FSC H and FSC A M ake sure the Current Tube pointer is next to the Unstained Control Tube Click Acquire View the events in the FSC H vs FSC A dot plot OH UF FP U N FF In the Instrument frame click the Laser tab 172 BD FACSCanto Flow Cytometer Reference M anual 8 Adjust FSC Area Scaling if necessary by clicking the arrows or moving the slider control Instrument arrows and Slider contro
29. to the next PM T Light with a longer wavelength will pass through to another LP filter 670 nm that further blocks shorter wavelength light Light from dyes such as PerCP Cy5 5 pass through this filter The beam continues around the array with the yellowish light from particles stained by fluorochromes such as PE emission spectrum 560 to 600 nm collected by PMT C and the green light from particles stained by fluorochromes such as FITC collected at PM T D PMT E collects side scatter signals blue laser light that was deflected by irregularities on the surface within the particles As you can see the arrangement of filters and mirrors allows each PMT to receive the majority of signals from a specific fluorochrome Unfortunately not all the screening can be done by filters and mirrors alone 160 BD FACSCanto Flow Cytometer Reference M anual Spillover Fluorochromes emit light over a range of wavelengths Asa result a signal from one fluorochrome can appear in a detector used for another fluorochrome Figure A 11 Figure A 11 Spillover of FITC into PE detector BP filters R3030 aiani f 100 ou BU nonrelized intensity 40 z 440 FOO 550 BOO 650 ro Wavelength nm emission spectrum of FITC FITC spillover into PE Emission spectrum of PE For example FITC appears primarily in the FITC detector but some of its fluorescence spills over into the PE detector PE appears primarily in the PE
30. 205060620 OZ020 O70200 O O00000 0 0 50000920920 O000000000 00000900909 O00000000000000900909 OKOH0CK0O0ZOZOKO 062992 OR O08 0599290020 o 05959 690K0 506069020 O27 020 020700 O 0696969050 506069020 080202056095 9620 o 2626262050 000009090 O00000000000000900909 ekte rda pdala hipiai 062902 OF 080899290020 060609650050 506069620 OxOxXO a p a ed P jE O 0596969050 506069020 0 020 apa a bal G D O O00000 0 0 50020920920 Okona oonan 5202090526 2926858059 020202069696 208050 060609609060 5060620202 02020 62020 0506060096 20 O 07 070 cytometer circuit breaker on If it is off turn the cytometer power on 2 Press the main power button to do this 124 BD FACSCanto Flow Cytometer Reference M anual Reconnecting the Ethernet and Network Cables The cytometer connects to and communicates with the workstation through an Ethernet cable and a network cable If these cables should become disconnected use the following diagram to reconnect them Ethernet cable network cable Both cables connect to ports on the PC workstation As the make and model of the workstation might vary refer to the documentation that came with your system for information Reconnecting the Fluidics Cart Tubing Should any plugs cords or tubings become accidentally disconnected use the following diagrams to reconnect the fluidics cart to the cytometer Figure 4 3 on page 126 M Tip The ports and connectors are colo
31. 3 7 100 881 29 823 29 666 3 014 59 93 14 M T cells 6 039 70 8 82 899 20 344 29 733 7 361 139 50 320 3 392 E T Helper 4 639 54 4 81 702 19 264 28 556 363 160 52 514 4414 BB T cytotoxic 1 312 15 4 86 586 23 853 33 525 33 069 7g 41 671 75 Chapter 3 Running Samples 89 Reusing the Analysis N ow that the analysis strategy has been defined you can use it to analyze the remaining Tubes in the Experiment Global worksheets allow you to apply an analysis strategy to a series of data files without saving the analysis each time After previewing the data you can print the analysis or save it to a Tubespecific worksheet see the following section Saving the Analysis 1 Movethe Current Tube pointer to the next Tube under the LWB Specimen 2 View thedata on the global worksheet make adjustments to gates as needed Adjustments will also apply to the next Tube that is viewed on the global worksheet If you don t want to alter the global worksheet save the analysis as described in the next section and make adjustments on the Tube s worksheet Saving the Analysis Since the Analysis objects were created on a global worksheet the analysis will not be saved with each Tube If you want to save a copy of the analysis with any Tube do the following 1 Expand the TBNK global worksheet in the Browser 2 Right click the Analysis object and choose Copy 4 6 Color Expt 2 Instr Settings Global Worksheets TENK Analysis la Analysis
32. 33 nm laser 750 810 nm APC Cy7 e 650 670 nm APC Photodiode with 488 10 bandpass filter PM T with 488 10 bandpass filter 182 BD FACSCanto Flow Cytometer Reference M anual Fluidics General operation Sheath consumption Sheath pressure Sample flow rates Sample acquisition rate Recommended maximum particle size Signal Processing Workstation resolution Data acquisition channels Fluorescence compensation Pulse processing Time Channel threshold Integrated fluidics cart with automated startup shutdown and cleaning cycles 1 08 L hr normal operation lt 1 0 mL hr standby 5 psi Reagent dependent controlled automatically by BD FACSCanto clinical software Low 10 uL min M edium 60 uL min High 120 uL min 10 000 events sec 50 um 262 144 channel resolution 8 parameters 6 fluorescent and 2 scatter parameters No limit to inter and intra beam compensation H eight Area and Width measurements available for any parameter BD FACSDiva software Can be correlated to any parameter Available for any parameter from all lasers Appendix D Technical Specifications 183 Fluidics Cart Specifications 184 Dimensions O perational clearances Weight Power Facilities Capacity BD FACSFlow cubitainer BD FACS cleaning solution cubitainer BD FACS shutdown solution cubitainer Waste tank H eight 66 cm 26 in Width 81 3 cm 32 in Depth 66 cm 26 in
33. 6 positive population on the CD3 APC vs CD16 56 PE plot name the population NK Cells 10 Draw aregion around the double positive population on the CD3 APC vs CD4 APC Cy7 plot and name the population T H elper 11 Draw aregion around the double positive population on the CD3 APC vs CD8 PerCP Cy5 5 plot name the population T Cytotoxic 88 BD FACSCanto Flow Cytometer Reference M anual 12 Print the analysis Figure 3 6 Figure 3 6 Lymphocyte analysis example LVVB TBNK_001 LVVB TBNK_001 NK Cells B Cells CD19 PE Cy amp T Cells j 10 104 10 104 107 10 104 CD45 FITC A CD3 APC A CD3 APC A LYVB TBNK_001 LYWB TBNK_001 10 T Cytotoxic 104 T Helper CD4 APC Cy A CD8 PerCP Cy5 5 A 10 107 a 10 104 10 10 CD3 APC A CD3 APC A Tube TBNK_001 Population Events Parent Total Hi All Events 30 000 100 0 LT Lymphocytes 8 535 28 4 28 4 B Cells 374 4 4 1 2 NK Cells 2 025 23 7 6 8 T Cells 6 039 70 8 20 1 T Helper 4639 54 4 15 5 T Cytotoxic 1 312 15 4 4 4 Experiment Name Immunophenotype Specimen Name LNB Tube Name TBNK_001 Record Date Aug 9 2004 6 39 18 PM OP Administrator FSC H S8C A CD45 FITC ACD16 56 P CD8 PerCP CD19 PE C CD3APC A CD4APC Population Events Parent Mean Mean Mean Mean Mean Mean Mean Mean LI Lymphocytes 8 535 28 4 87 756 22 800 29 539 2 036 5 938 1 096 35 638 2 415 B e Cells 374 4 4 85 999 21 558 27 181 17 5 22 434 73 27 E nk cells 2 025 2
34. A 7 Longpass filter longpass 480 520 460 500 540 transmission wavelength nm N ot all light with shorter wavelengths is absorbed or reflected Some will still pass through Bandpass Filter A BP filter transmits 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 efficiently between 475 and 525 nm Figure A 8 Appendix A Technical Overview 15 7 Figure A 8 Bandpass filter bandpass 460 500 540 t29 480 520 C O A f 50 X 0 450 500 550 wavelength nm Detectors Detectors convert light signals into electrical signals that can be processed by the electronics system and a computer and then displayed on a plot There are two types of signal detectors in the BD FACSCanto flow cytometer the photodiode and the photomultiplier tube PM T The photodiode Is used to detect the stronger FSC signal generated by light from the blue laser The more sensitive PM Ts are used to detect the weaker signals generated by SSC and all fluorescence channels In BD FACSCanto clinical software the fluorochromes are preset In BD FACSDiva software the Instrument Configuration dialog box lets you define the f
35. BD FACSCanto Flow Cytometer Reference Manual For In Vitro Diagnostic Use http www bdbiosciences com Part No 338617 Rev A September 2004 BD Biosciences Asia Pacific 2350 Qume Drive Tel 65 6 861 0633 San Jose CA 95131 1807 Fax 65 6 860 1590 USA Tel 877 232 8995 Fax 408 954 2347 Europe Tel 32 53 720211 Fax 32 53 720452 Brazil Tel 55 11 5185 9995 Fax 55 11 5185 9895 Japan Nippon Becton Dickinson Company Ltd Tel 0120 8555 90 NC Canada Tel 888 259 0187 905 542 8028 Fax 905 542 9391 canada bd com Mexico Tel 52 55 5999 8296 Fax 52 55 5999 8288 2004 Becton Dickinson and Company All rights reserved No part of this publication may be reproduced transmitted transcribed stored in retrieval systems or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without prior written permission from BD Biosciences The information in this guide is subject to change without notice BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments Although this guide has been prepared with every precaution to ensure accuracy BD Biosciences assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information BD Biosciences welcomes customer input on correcti
36. FACS 7 color setup beads refer to the reagent instructions for use 2 Prepare the cytometer for manual loading Se a adapter lever in highest position Y tube guide pushed back aspirator arm bar vertical drawer pulled out carousel removed 3 Choose Cytometer gt Setup Cytometer Standby Shutdown Fluidics Startup Fluidics Shutdown Cleaning Modes The Cytometer Setup Wizard appears 62 BD FACSCanto Flow Cytometer Reference M anual 4 Select the current bead lot from the Lot ID drop down menu and click Cytometer Setup Wizard Setup Lot Information Bead Lot Information Select the lot information for your bead product Lot ID 8675309 Bead Product BD FACS 7 Color Setup Beads 335775 Exp Date 2005 12 31 New Lot ID Targets Spectral Overlap Factors Scatter Fluorophore Target Value AESC SSC FITC PE PerCP PerCP Cy5 5 PE Cy APC APC Cy B Cancel Refer to the BD FACSCanto Clinical Software U ser s G uide for information on entering new Lot IDs Targets and Spectral O verlap Factors 5 Select Run setup in Manual mode and click new gt _ Cytometer Setup Wizard Insert Setup Tube Insert Setup Tube Insert the bead tube Lot ID 8675309 click Next to begin setup Run setup in Manual mode Chapter 3 Running Samples 63 6 When prompted load the bead tube onto the S
37. FACSFlow port waste port gt FACSFlow waste tank BD FACSFlow cubitainer Each solution has its own non interchangeable fluid port and level sensor connection Fluid level alarms occur within BD FAC SCanto clinical software and BD FACSDiva software Chapter 1 Introduction 37 38 Controls The fluidics cart connects directly to the flow cytometer unit via a power cord When you turn on the power to the cytometer the fluidics cart powers on also Under ordinary circumstances you do not need to adjust any of the switches on the cart s power panel located on the side of the cart Leave the auxiliary air supply switch off as shown in Figure 1 9 unless the cart has been attached to an in house air supply by BD Biosciences service personnel and leave the cart circuit breaker on at all times Figure 1 9 Fluidics cart panel auxiliary air suppl Sitch Off ii Auxiliary Air Supply cart circuit breaker on Powering Off To turn off the fluidics cart and the cytometer as well press the cytometer main power button During cart shutdown you will normally hear a hiss and a small amount of condensed water will discharge from the pumps BD FACSCanto Flow Cytometer Reference M anual Computer Workstation The workstation consists of a computer compatible with M icrosoft Windows X P Professional operating system a flat screen monitor and a printer
38. IT BD FACSCanto A Please load the setup tube i Cancel e Push the aspirator arm to the left e Place the beads tube onto the SIT and push up until the tube is firmly seated e Center the aspirator arm under the beads tube 7 Click 8 Wait for setup to finish Cytometer Setup Wizard Running Setup Setup Status Please wait while cytometer setup executes symbols key Tasks completed Placing beads on scale gt Characterizing instrument ps in process E ti wp enerating setup Indone Event Rate 904 64 BD FACSCanto Flow Cytometer Reference M anual 9 Unload the bead tube when prompted Unload Tube t Please unload the setup tube e Hold the sample tube while pushing the aspirator arm all the way to the left e Remove the tube from the SIT e Release the aspirator arm SIT cleaning occurs when the aspirator arm comes to center 10 Optional View the Setup report by clicking __ view Setup Repot 11 if setup is successful click Finish Chapter 3 RunningSamples 65 66 12 If setup completed but some parameters were out of range decide how you want to proceed Cytometer Setup Wizard Setup Complete Setup Completed Successfully Click Next to optimize Setup Tasks Completed Setup completed Some parameters were out of specification Click Next to optimize view Setup Report Discard current You will be given the option to results use the last setup resul
39. Loader Optional The Loader automatically introduces prepared samples to the cytometer It consists of a drawer a cover two optical sensors an electronics module a tube lifter and a 40 tube carousel O perate the Loader from within either BD FACSCanto clinical software or BD FACSDiva software You can add the Loader to your system at any time Refer to the BD FACSCanto O ptions M anual for information about the Loader Chapter 1 Introduction 39 System Requirements 40 Software BD FACSDiva or BD FACSCanto clinical software depending upon your laboratory needs Workstation BD FACSCanto workstation purchased through BD Biosciences Tubes e 12 x 75 mm polystyrene BD Falcon tubes e 12x 75 mm BD Trucount tubes Bulk Fluids e BD FACSFlow solution e BD FACS cleaning solution e BD FACS shutdown solution e full strength bleach waste tank Other Fluids Required for External Cleaning e BD FACS cleaning solution e Deionized DI water Setup Beads BD FACS 7 color setup beads and tubes for use with BD FACSCanto clinical software BD FACSCanto Flow Cytometer Reference M anual 2 Using BD FACSDiva Software You can control most BD FACSCanto instrument functions using either BD FACSCanto clinical software or BD FACSDiva software BD FACSCanto clinical software contains modules for dedicated clinical applications with automatic gating algorithms while BD FACSDiva software is non application spec
40. Octagon and trigon detector arrays red laser signal NI octagon J longpass dichroic y mirror CE bandpass D filter blue laser signal BD FACSCanto Flow Cytometer Reference M anual When light arrives at an array a long pass dichroic mirror transmits the highest wavelengths to the first PM T in the series and reflects lower wavelengths to the next PM T Likewise the next PM T s long pass dichroic mirror will transmit the next highest wavelengths and reflect lower wavelengths and so on around the array A bandpass filter in front of each PM T further screens unwanted light Fiber In addition to the PM T detectors a photodiode collects the stronger forward scatter signals The obscuration bar prevents excess laser light from entering this diode Figure 1 3 on page 29 O ptics System on page 153 further discusses the detector arrays and how dichroic mirrors and filters work Chapter 1 Introduction 33 At installation the octagon and trigon arrays have the filter and mirror combinations shown in Table 1 1 Table 1 1 Octagon and trigon optical filters Detector Array PMT LP Mirror BP Filter or LP Mirror Intended Laser Position Dye Octagon A 735 780 60 PE Cy7 488 nm blue laser 655 670 LP PerC P Cy5 5 PerCP C 556 585 42 PE D 502 530 30 FITC E blank 488 10 Side scatter optical holder SSC F blank blank optical holder optical holder G blank blank optic
41. When using BD FACSCanto clinical software the cytometer automatically regulates the sample pressure according to the currently selected panel Optics System As stained cells or other particles pass through the focused laser beam they scatter the laser light and fluoresce Because the laser beam is focused to 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 This brief flash of light is collected filtered and then converted into an electrical signal by the detectors Figure A 3 Figure A 3 Light pathway through the BD FACSCanto flow cytometer stained particle fiber optic cables prisms focusing collecting forward scatter lens lens diode detector C I z ANH S f obscuration steering plate as i fow cel bar LP red laser mirror fiber optic BP S cable filter blue laser PMT detector The following sections discuss these processes in greater detail Appendix A Technical Overview 153 Light Scatter When a cell or particle passes through a focused laser beam laser light is scattered in all directions Figure A 4 Light that scatters roughly in the same direction as the laser beam is called forward scatter FSC light that scatters roughly perpendicular to the laser beam Is called side scatter SSC FSC and SSC intensities are related to certain physical properties of cells e FSC indicates
42. afe wicr ievi eo aks oad eb crea Ss Bee eh eee dR ae XIX Po Set CMON AUS sareei Ri a aca as Sak an ahaal ateaa eas era oe Sy ish a aad e Mitac Shae at XX EIN CGE ONG ap sce shea td Scdeagiene vireo gat deat ata wee av aw ee Se alae ol cg eee Bee aa xxii BD FACSDiva Software Limitations saana aaaea xxiii Chapter 1 Introduction 25 intended USC s oct ach ee teaetechadee na ea ae eaa a i a a 26 System COMPONENTS ceanna a E a a T A eat 26 CHOME vial Gad dh eae an cry et a eed aed a 27 PIUIGICS Cart gag i ctowitnte 8 wien ET e TEE EEEE 36 Computer Workstation 0 ccc eee eee ees 39 Loader Optional ncites vee FAS eeri i ale ee ee es 39 SVStCM CCU EmMEN Se sc 5 8 rat atts Fuca cy sone a eke bard aes Chapter 2 Using BD FACSDiva Software Workspace Components 0 0 0 cc eee eee eee Instrument Menu Commands 0c eee ete eee FUNGI GS CONTOS erine cache a be s aeulse a At as ae eat ee Instrument Configuration 0 cee eee ees Instrument Status Report ce et ees wnd Yess rearea a Ate ane ele oer eee atin ae Controls in the Instrument Frame 0 0 ee ees Fluid Level Indicators cee eee tees LASER Waist aeraceeeiivaara neee ay in ete saw tea ie bac Gl Ose sates Geta aa ath a coal ese ee greed ae ae pt deen nih eet ACQUISITION CONOIS 3 tbh atatdakdod eE ee wl eae es Chapter 3 Running Samples WORKTIOW tx sates ba bare ee ale eek BAe are ae INSEPUIMOCNC StQRIUD ca sib ot dort be doh ara ao
43. al holder optical holder H blank blank optical holder optical holder Trigon A 735 780 60 APC Cy7 633 nm red laser blank 660 20 APC Blank optical holders do not contain any glass in the central opening They are used in the octagon and trigon to prevent unwanted light from interfering with your fluorescence signal 34 BD FACSCanto Flow Cytometer Reference M anual Electronics The electronics system converts optical signals to electronic signals and digitizes them for computer analysis The photodiode and PM Ts generate signals proportional to the amount of light they receive The cytometer s onboard electronics amplifies and then converts the signals from continuous voltage values analog into discrete values digital Upon amplification and digital conversion fluorescent light signals from consistently prepared and stained particles characteristically fall into certain channels thus allowing analysis On the BD FACSCanto electronic system components consist of power controls and connectors along with processing boards in the card cage This section describes only user adjustable instrument electronics For more information see Electronics System on page 163 Power Panel Power to the instrument lasers and fluidics cart is supplied by a power cord from the cytometer plugged directly into a standard electrical outlet The main power button turns on the instrument and fluidics cart and powers the lasers Figure 1 6 Flo
44. al instrument functioning perform the following maintenance according to the recommended schedule Table 4 1 Scheduled Maintenance Maintenance Procedure Daily Startup e For BD FACSDiva software see Instrument Startup on page 57 e For BD FACSCanto clinical software refer to Daily Startup in the BD FACSCanto Clinical Software User s Guide Daily Shutdown e For BD FACSDiva software see Daily Shutdown on page 92 e For BD FACSCanto clinical software refer to Daily Shutdown in the BD FACSCanto Clinical Software User s Guide Changing a Cubitainer on page 98 Priming on page 101 Emptying the Waste Container on page 102 Changing the Waste Tank Cap see step 6 on page 104 Purging the Fluidics Filters on page 105 Decontaminating the Fluidics System Long Clean on page 107 Recommended Frequency Every day Every day Check fluid levels daily and as needed Whenever a fluidics line is disconnected to change a cubitainer or perform other maintenance Check waste level daily and as needed Every month Every week Every month BD FACSCanto Flow Cytometer Reference M anual Table 4 1 Scheduled Maintenance continued Maintenance Procedure Recommended Frequency Replacing the Air Filter on page 108 BD Biosciences recommends replacement every 6 months Replacing Fluidics Filters on page 111 BD Biosciences recommends replacement every 6 months Chapter 4 Maintenance 97 Changing a Cu
45. alue remains valid whether a dim or bright sample is run Figure A 14 demonstrates this principle Although the signals differ in intensity the percentage of signal detected in the FITC and PE channels remains constant Figure A 14 Two FITC signals of different intensity FITC PE different intensity FITC signals nonrelized intensity same proportion or percentage of spillover in PE channel 500 nm S50 Arm 600 nm 650 nm 700 nm Electronics System Any discussion of electronics requires a basic understanding of the bit Computers and digital circuits use bits binary numbers consisting of ones and zeros to pass information along A 4 bit number has 4 digits that are either 1 or 0 A 10 bit number has 10 digits that are either 1 or 0 An 18 bit number has 18 digits that are either 1 or 0 000000000000011110 is one example of an 18 bit number Converted into base 10 the scale we normally use this number Is equivalent to 30 All together there are 262 144 possible 18 bit numbers Appendix A Technical Overview 163 Pulses Inside the PM Ts the laser light is converted into an electrical signal This electrical signal is called a pulse Figure A 15 Figure A 15 Anatomy of a pulse ee j SS ie g particle T time i b a E g A time i ies g A 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 maxim
46. ary air supply switch Keep in off position unless connected to house air i Fluid Out BD FACSFlow solution port j Communication Data port k Power In Connects to cytometer Do not connect to wall outlet auxiliary air in There will be no tubing on this port unless connected to house air m Air Out Sends compressed air to cytometer n Waste Waste in o Waste Waste in under vacuum Chapter 4 M aintenance 127 Removing an Air Lock If too much air gets into the sheath filter it becomes impermeable to fluid and an air lock can develop To removean air lock follow this procedure Materials e paper towels e bypass tubing Procedure 1 Place a few paper towels beneath the air locked filter to collect drips 2 Remove the filter by pressing the tabs on each quick disconnect coupling metal tab quick disconnect coupling metal tab 128 BD FACSCanto Flow Cytometer Reference M anual 3 Install the bypass tubing in place of the filter Push the tubing into each quick disconnect port until you hear a click bypass tubing installed 4 Choose Instrument or Cytometer gt Cleaning M odes gt Prime After Tank Refill 5 Select the checkbox that corresponds to the filter you changed click OK Prime After Tank Refill x Select tanks to prime FACSFlow Boia selden Please select the checkboxes for the tanks that need to be primed Cleaning solution FACSFlow Shutdown Solu
47. as been recorded you need to adjust the gates around the positive populations on the histogram for each stained control Tube 1 Doubleclick the first Stained Control Tube FITC Stained Control to locate the corresponding plots on the worksheet 80 BD FACSCanto Flow Cytometer Reference M anual 2 If needed move the P2 gate to encompass the fluorescence positive population Figure 3 5 Gating the positive populations FITC Stained Control 3 Doubleclick the next Stained Control Tube in the Browser to locate the corresponding plots on the worksheet 4 Repeat steps 2 and 3 for the remaining compensation Tubes Creating a Compensation Matrix Once all gates have been adjusted you are ready to calculate compensation 1 Choose Instrument gt Instrument Setup gt Calculate Compensation If the calculation is successful a dialog box appears where you can enter a name for the compensation Setup 2 Enter a name for the compensation Setup click OK Single Stained Setup Compensation calculation has completed successtully Mame 6 Color Expt 030503 cs Vv Tip To keep track of compensation Setups include the Experiment name date or both in the Setup name Chapter 3 Running Samples 81 The named Setup is automatically linked to the Experiment s instrument settings M Tip Collapse the Compensation Specimen to save room in the Browser BD Biosciences recommends that you confirm the compensation setup by running a
48. atents PE and APC US 4 520 110 4 859 582 5 055 556 Europe 76 695 Canada 1 179 942 PerCP US 4 876 190 Cy5 5 and Cy7 US 5 268 486 5 486 616 5 569 587 5 569 766 and 5 627 027 Pe Cy7 US 4 542 104 APC Cy7 US 5 714 386 FCC Information WARNING Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment NOTICE This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual can cause harmful interference to radio communications O peration 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 C ausing Equipment R egulations Cet appareil num rique dela classe A respecte toutes les exigences du R glement sur the mat riel brouilleur du Canada Notice BD Biosciences delivers software
49. bitainer Three fluidics cubitainers disposable boxes of approved fluids and a waste container fit onto the cart in the following configuration BD FACS shutdown solution BD FACS cleaning solution FACSFlow a q7 waste container BD FACSFlow solution Each cubitainer and the waste tank has its own port Connect the cubitainers and waste tank to the ports listed in the following table BD FACSFlow solution BD FACSFlow BD FACS shutdown solution BD FACS Shutdown Solution BD FACS cleaning solution BD FACS Cleaning Solution Vv Tip The ports and connectors are color coded 98 BD FACSCanto Flow Cytometer Reference M anual Procedure 1 Ensure that the cytometer is not acquiring events 2 Detach the sensor and fluid line from the cart e To detach the sensor turn the connector counterclockwise and pull sensor a i a en A D FACS Cleaning i N 3 n i T STORA RR i e To detach the fluid line press the metal clip on the quick disconnect coupling metal clip A You could damage the sensor line if you leave it connected while changing a cubitainer Chapter 4 Maintenance 99 3 Unscrew the cap on the cubitainer 4 Removethecap assembly and set it aside Figure 4 1 Cubitainer cap and sensor The cap sensor and fluid lines and the level sensor come attached in a single assembly piece 5 Put anew cubitainer onto the fluidics cart 6 Replace th
50. d sample loader is also available Figure 1 1 BD FACSCanto system For a description of system components see the following sections e Cytometer on page 27 e Fluidics Cart on page 36 e Computer Workstation on page 39 e Loader Optional on page 39 26 BD FACSCanto Flow Cytometer Reference M anual Cytometer W ith the exception of the power button you control all cytometer and fluidics cart functions from within the two software packages provided with the instrument BD FACSCanto clinical software and BD FACSDiIva software BDFACSCanto flow cell access door 4 side door data ports optics access door OBDFACSCanto sample injection tube power button fluidics cart connections The BD FACSCanto flow cytometer consists of an optics subsystem a fluidics subsystem and an electronics subsystem For a more in depth discussion of fluidics optics electronics and flow cytometry see the Technical Overview on page 149 Fluidics The fluidics system consists of the sample injection tube SIT the aspirator arm the flow cell a pressurized interior reservoir and a network of tubing that provides sheath and cleaning fluids to and removes waste from the flow cell See Figure 1 2 on page 28 Chapter 1 Introduction 27 Figure 1 2 Sample injecti
51. detector but some of its fluorescence spills over into the FITC detector This spillover must be corrected or compensated for Thus the term compensation In Figure A 11 a portion of the FITC emission will be detected by both the FITC and PE channels This can be seen in an x y plot of FITC vs PE Figure A 12 on page 162 In this figure the green circle represents a population stained with FITC the gray circle represents a population negative for both FITC and PE Appendix A Technical Overview 161 Figure A 12 Theoretical display of FITC vs PE before compensation PE If the fluorescence is to be assigned to PE the FITC signal must be removed from the PE channel as indicated by the arrow Both PE and FITC fluoresce in the yellow 575 nm range so there is no way to Isolate the emission from each fluorochrome optically Instead fluorescence compensation moves the FITC population out of the PE positive area Figure A 13 Figure A 13 FITC signal properly compensated out of the PE channel PE FITC The software automatically computes these adjustments during setup which you can further refine e In BD FACSDiva software adjust spectral overlap in the Compensation tab in the Instrument frame e In BD FACSCanto clinical software adjust spectral overlap on the Spectral Overlap tab 162 BD FACSCanto Flow Cytometer Reference M anual Once compensation has been set for one sample the spectral overlap or compensation v
52. e Black represents the amount of empty space in a tank e Red indicates that a tank needs service because it is full waste tank or empty fluid tanks Vv Tip Place your cursor over one of the boxes to see which fluid level it represents 48 BD FACSCanto Flow Cytometer Reference M anual When BD FACSFlow solution is low lt 17 or the waste Is nearly full gt 83 during acquisition a message such as the following displays 2 Waste is full please empty Close If you e close the message box without replacing the BD FACSFlow container or emptying the waste the message reappears every 5 minutes The system continues running e do not close the dialog the fluidics will shut down in 15 minutes If the BD FACSFlow container becomes empty 0 or the waste becomes full 99 the system shuts down You will need to service the indicated container to continue Chapter 2 Using BD FACSD iva Software 49 50 Laser Tab The values in the Laser tab compare measured laser current or power to a reference point set during instrument installation If the measured value is 20 higher or lower than the reference value an error message appears in the Status tab Figure 2 6 When this occurs make sure the flow cell access door Is completely closed If this does not resolve the problem call your BD Biosciences service representative for assistance Figure 2 6 Laser tab Blue Laser Current Blue Laser Power Red Lase
53. e cap assembly and hand tighten it until it is fully closed 7 Reattach the sensor line and fluid line to the cart e To attach the sensor line gently rotate until the connection aligns and then push e To attach the fluid line push the quick disconnect coupling into the port until it clicks into place A To ensure that the appropriate solutions are dispensed match the label on the container to the port on the fluidics cart 100 BD FACSCanto Flow Cytometer Reference M anual 8 Prime the fluidics IMPORTANT Go to Priming Priming Use the Prime After Tank Refill command to remove air from the fluid lines after you change a cubitainer or detach the fluidics lines for other maintenance A The BD FACSDiva Instrument menu corresponds closely with the BD FACSCanto Cytometer menu There are slight discrepancies between the menus for example there are more commands in the BD FACSDiva Instrument menu Instructions in this and following sections list the BD FACSDiva menu first followed by the BD FACSCanto menu 1 Choose Instrument or Cytometer gt Cleaning M odes gt Prime After Tank R ef ill 2 Select the checkboxes for the cubitainers you changed click OK Prime After Tank Refill Select tanks to prime FACSFlow FJ Tank Prime Shutdown solution Please selectthe checkboxes for the tanks that need to be primed Cleaning solution FACSFlow Shutdown Solution Cleaning Solution l cancel BD FACSDiva soft
54. e oo das ale hae aoe Instrument Quality Control cc ce ee Performing Automated Setup nananana eea Optimizing Instrument Settings 0 0 cee ee ees Data Recording and Analysis 00 c eee eee Setting Up the Global Worksheet 0 00 e ee eee Recording Dala ee eosctote wince wont dt aoa oe Ao ead a hae bo ea eae Analyzing Data oak sich adcactinct a a am ete Reusing theAnalysis 0 cc eee tees Sa VITO CMG HAGNY SIS rarman aaa a Sew daw ate aes EJJIN OUE errre Aca ie ence 4G nine ana ene ee Dally SNULGOWN lt s3 cniuta el teorw ne PAGO a ad Sea waa saa Chapter 4 Maintenance Scheduled Maintenance ccc ee eee eas vi BD FACSCanto Flow Cytometer Reference M anual 40 41 42 43 43 44 46 47 48 48 50 51 52 55 56 57 61 61 68 83 83 85 87 90 90 91 92 95 96 Channa CU DILGIN Cl 35 5s iat attest ati eatel aon Sey ah ana ahe de yee ae 98 Emptying the Waste Container 0 cc te ees 102 Purging the Fluidics Filters 0 0 ccc eee ees 105 D econtaminating the Fluidics System Long Clean 5 107 Replacing the Alr Filter sasscnadsedkeedea dant aas daa nawolea oleae 108 Replacing Fluidics Filters nnna ccc ee ee eee teens 111 Unscheduled Maintenance 1 0 cc ee ete 114 Removing Bubbles from the Flow Cell 0 cee eee eee 115 Cleaning the Flow Cell ce eens 116 Purging the Bubble Filter rinri riaraitt es 117 Cleaning External Sur
55. e subset You must create manual gates when using BD FACSDiva software The software does not provide application specific analysis templates Safety and Limitations xxiii xxiv BD FACSCanto Flow Cytometer Reference M anual 1 Introduction The BD FACSCanto system sets a new standard for performance in flow cytometry With its fixed optics design and digital electronics the BD FACSCanto flow cytometer allows multicolor analysis of up to six fluorescent markers and two scatter parameters at a time You need no special facilities the instrument plugs into a standard wall outlet uses air cooled lasers and provides its own air pressure and vacuum for the fluidics and waste You can prepare samples on the BD FACS Sample Prep Assistant II and import the worklist For further automation use BD FACSCanto clinical software and the BD FACS Loader for sample acquisition You can also use BD FACSDiva software for more flexibility in acquisition and analysis This chapter contains these topics e Intended Use on page 26 e System Components on page 26 e System Requirements on page 40 25 Intended Use The BD FACSCanto flow cytometer identifies and enumerates lymphocyte subsets in human cells in suspension System Components The BD FACSCanto system consists of three major components a benchtop flow cytometer a self contained fluidics cart and the BD FACSCanto computer workstation see Figure 1 1 An optional automate
56. e to follow this sequence could result in carryover between samples e Click Remove Tube A progress dialog appears e Hold your sample tube with one hand while you push the aspirator arm all the way to the left with the other hand Always hold onto your sample tube when you push the aspirator arm to the side If you move the arm without holding the tube the tube could fall off the SIT and expose you to potentially blohazardous sample e Remove the tube from the SIT e Release the aspirator arm SIT cleaning occurs when the aspirator arm comes to center Progress YD Remove tube in progress Do notload next tube until flush is complete 6 When theProgress dialog disappears you can load the next tube onto the SIT 86 BD FACSCanto Flow Cytometer Reference M anual 10 M ove the pointer to the corresponding Tube in the Browser and click Acquire Preview the data in the global worksheet click Record to record data Repeat steps 5 through 8 until data has been recorded for all tubes O ptional Print the Experiment level instrument settings or the Instrument Status report To print the settings right click the instrument settings icon and choose Print 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 3 6 on page 89 1 2 Create a new global workshe
57. eall biological specimens and materials as if capable of transmitting infection Dispose of waste using proper precautions and in accordance with local regulations N ever pipette by mouth Wear suitable protective clothing eyewear and gloves e Always wear gloves when manually loading samples A fluid flush of the exterior of the sample injection tube SIT occurs between samples that might contain biohazardous waste e To prevent a biohazardous spill during manual loading always hold the Sample tube on the SIT when you move the aspirator arm e Expose waste container contents to bleach 10 of total volume before disposal Dispose of waste in accordance with local regulations Use proper precaution and wear suitable protective clothing eyewear and gloves e Prevent waste overflow by emptying the waste container daily or whenever the waste indicator in the software shows the waste is getting full e Thewaste tank can become pressurized when the cytometer is running Always disconnect the tank from the fluidics cart before you empty it Wait at least 30 seconds for pressure to dissipate before you remove the waste Cap or sensor e Replace the waste tank cap every month Failure to do so could cause the waste tank to malfunction For tracking write the date on the waste cap label each time you change the cap xvi BD FACSCanto Flow Cytometer Reference M anual e Do not wet the waste tank cap If wet the filter in the cap will cau
58. eneral optimization of instrument settings consists of the following steps each step is explained in detail in the sections that follow It is important that you perform these steps in order You might need to vary certain steps for different sample types e Verify Instrument Configuration and the User Preferences are set appropriately e Create an Experiment e Apply the BD FACSDiva software instrument settings to the setup generated by BD FACSCanto clinical software e Optimize instrument settings e Calculate compensation 68 BD FACSCanto Flow Cytometer Reference M anual Verifying Instrument Configuration and User Preferences Check the instrument configuration and User Preferences before you set up your Experiment 1 Choose Instrument gt Instrument Configuration and verify that the current configuration contains appropriate parameters 2 Verify that the filters are appropriate to run FITC PE PerCP or PerCP Cy5 5 PE Cy7 APC and APC Cy7 fluorochromes A For accurate data results the octagon and trigon arrays must match the current Instrument Configuration The default filter configuration is appropriate for six color assays 3 Choose Edit gt User Preferences and select the following values User Preferences General Gates Plot Fcs Templates Carousel Tube specitic worksheet Start acquisition on pointer change IV Remove tube specific instrument settings on duplicate iV Save analysis a
59. et rename the worksheet TBN K Analysis Select the first Tube under the LW B Specimen and create the following plots on the Analysis template e FITC vs SSC e APC vs PE Cy7 e APC vs PE e APC vs APC Cy7 e APC vs PerCP Cy5 5 Resize the plots so they fit on one page as shown in Figure 3 6 on page 89 On the FITC vs SSC plot draw a gate around the lymphocytes use the Population Hierarchy to rename the population Lymphocytes Chapter 3 RunningSamples 87 5 Select all plots except the FITC vs SSC plot and specify to show only the Lymphocyte population H old down the Control key while you select successive plots O nce all plots are selected click the checkbox next to Lymphocytes in the Plot Inspector 6 Select all plots and click the Title tab in the Plot Inspector select the checkboxes to display the Tube and Specimen names in the plot titles inspector Plot Title Labels Dot Plot Title Content V Tube Populations v Specimen 7 Create a Statistics view edit the view to show the Lymphocyte population and subpopulations and to display the mean for all fluorochromes For instructions on how to create and edit a Statistics view see the BD FACSD iva Software Reference M anual 8 Draw aregion around the CD19 positive population on the CD3 APC vs CD19 PE Cy 7 plot name the population B Cells Vv Tip When drawing regions be sure to include events on the plot axes 9 Draw aregion around the CD16 5
60. events for Tubes TBN K_001 and TBN K_002 click OK Notice that the Acq tab in the Inspector updates automatically Experiment Layout Labels Keywords Acquisition Events to Record 10 000 v 6 Color Expt oO We 10 U TBNK_001 iat 10000 U TBNK_002 5 On theglobal worksheet create appropriate plots for previewing the data For example create FITC vs SSC APC vs PE Cy7 APC vs PE APC vs APC Cy 7 and APC vs PerCP Cy5 5 dot plots M Tip Double click the Plot tool to keep the tool selected until you create all plots Click any other button to undo the selection Recording Data If the Save Analysis After Recording preference is enabled in User Preferences a copy of the data displayed in the global worksheet will be saved with each recorded Tube If you do not want to save a copy of the data disable the preference 1 install the first sample tube onto the cytometer 2 Movethe Current Tube pointer to the first Tube click Acquire A Do not place any heavy objects on top of the cytometer at any time doing so could cause alteration of data Chapter 3 Running Samples 85 3 While data is being acquired draw a gate around the lymphocytes set the other plots to show data from the Lymphocyte population 4 Click Record to record data 5 When all events have been recorded follow these instructions for tube removal A It is critical that you follow the tube removal sequence exactly Failur
61. faces ec eens 118 D econtaminating the Fluidics System for Storage 0005 119 Replacing the Bal Seal sw eeviadwas ha eee SAG ewe a es 120 Resetting the Cytometer Circuit Breaker nunua aana 124 Reconnecting the Ethernet and Network Cables 00085 125 Reconnecting the Fluidics Cart Tubing 00 cee eee 125 RemovinG al AIr LOCK erras prenia Kaa ew bo Rate ha ae Phe 128 Replacing the Fluidics Level Sensors cece ee ees 130 Replacing the Fluidics Cart FUSES 0 tees 134 Chapter 5 Troubleshooting 137 INStrument T EOUBIESIVC 0 GG aoites refine tiated dated Ge aha bees ah tea eee 138 Fluidics Cart Troubleshooting auushiade verde ue Geek each rele eee eS 142 Acquisition Troubleshooting for BD FACSDiva Software 0 0 ccc ee ee eens 143 Appendix A Technical Overview 149 PLOW CGO MICE sc see s ae cacids scat dpe des Geetha ack eld Roa eee aed Sew See it 150 FLU CH CS Syte aeree a Wee tp Ake ew ak Pa Ae wea ee a 150 ODUCS SV SCCM t o Hoard an eva ad a Ma Wend at Hee ace waka dad ard b oa aaa 153 LAGMESCAULCR amarae Heald Ee Ae tn E ead Gra Wade Bae A he 154 Contents vii FO ROSC ONG aig a 5 tie at we ch cata es ee eat wt Eos cast ak antag heh eink Gn wae oe 154 Optical I EGES crr iiem pa dia a dy antes ROD Re ae and Sed oe ara 155 LCCC sical hci sor sate de PW tees ish aace A tele seeds iran Se ee acne shy hE 158 DiGlCChON AT PAVS wiesiueete a s Swe ket esi al ae wah etek SySin whe w
62. fications 180 184 starting 57 status report 46 supplies 176 interlock laser path 31 isopropyl alcohol 118 L labels parameter 84 lasers current 50 described 30 interlock 31 power 35 Safety xvii shown 31 specifications 181 troubleshooting 141 leaks cytometer 140 fluidics cart 142 SIT 141 levels fluid 48 58 sensor 100 130 limitations system xx Loader 39 loading tubes 64 logging in 58 out 91 Long Clean 107 210 BD FACSCanto Flow Cytometer Reference M anual longpass LP filters 156 M maintenance scheduled 96 unscheduled 114 manual setup mode 63 menu Instrument 43 mode automatic to manual 121 standby 47 O obscuration bar 29 33 octagon optics 32 34 optics access door 27 components 30 dichroic mirrors 156 fiber 31 filters default 34 filters theory 155 optical pathway 31 optimization Instrument 61 Instrument settings 68 ordering supplies 175 P parameters assigning 44 labels 84 scatter distorted 145 photodiode 29 33 photomultiplier tubes PM Ts applying voltages 75 assigning 44 described 32 plots excessive debris 145 no eventsin 143 unexpected eventsin 144 populations troubleshooting 146 power button 27 35 fluidics cart 38 instrument 35 laser 35 preferences user 91 Prime After Tank Refill 101 priming fluid lines 101 prisms 31 purging filters 105 Q QC cytometer 61 quality control QC 61 log 201 performing 61 R recording compensation T
63. fter recording through global worksheet iV Automatically update instrument settings according to latest setup For a complete discussion of User Preferences refer to the BD FACSD iva Software Reference M anual Chapter 3 RunningSamples 69 Creating the Experiment This section describes how to create a folder and an Experiment specify the parameters for the assay and add compensation Tubes 1 Click the corresponding buttons in the Workspace toolbar to display the Browser Instrument Inspector 4 Worksheet a and Acquisition Controls 3 and Carousel Control amp frames as needed Vv Tip Asyou work in the software frames can become hidden Y ou can easily bring a frame to the forefront by double clicking the corresponding button in the Workspace toolbar 2 Optional Create a folder for your Experiment e Select your database icon in the Browser x Browser I Userhlame EHO Folder _001 EHE Experiment_001 6604 12 31 3 aS Instr Settings LHE Global Worksheets FH Global Sheet E s Specimen 001 database icon HHE Shared view e Click in the Browser toolbar rename the folder Immunophenotype 3 Select the folder icon and click to create a new Experiment An open Experiment appears open experiment closed experiment BB Experiment _006 i Experiment _006 tT Instr Settings Global Worksheet 70 BD FACSCanto Flow Cytometer Reference M a
64. g is needed Materials e 3mL of BD FACS cleaning solution e onel2 x 75 mm polystyrene BD Falcon tube Procedure 1 Choose Instrument or Cytometer gt Cleaning M odes gt Clean Flow Cell 2 If you havea Loader remove the carousel 3 When prompted manually install a tube containing approximately 3 mL of 10 bleach onto the SIT and click OK Gean Fow Cell Install a tube with cleaning solution on A Please remove the carousel and install a tube with cleaning solution Click OK the SIT and click OK BD FACSDiva software BD FACSCanto clinical software The instrument cleans the flow cell A progress message appears during the cleaning Figure 4 2 on page 117 116 BD FACSCanto Flow Cytometer Reference M anual Figure 4 2 Clean Flow Cell progress message Cleaning Flowcell Please wait while the Flowcell is cleaned Progress YD Clean Flow Cellin progress please wait We BD FACSDiva software BD FACSCanto clinical software 4 After the completion message appears wait 5 minutes Waiting allows the BD FACS cleaning solution to dissolve deposits in the flow cell cuvette 5 Click OK 6 Remove the tube from the SIT 7 Run Fluidics Startup to remove BD FACS cleaning solution from the line Purging the Bubble Filter The bubble filter sits between the fluidics tanks and reservoirs and the flow cell and ensures that the flow cell remains bubble free H owever should the fluidics run dry you might need t
65. he waste tank Positive pressure pumps In the cart send sheath past a 0 22 um filter to a pressurized interior reservoir within the cytometer called the plenum The plenum maintains a nearly constant fluid level and dampens pump pulsation As a result sheath pressure does not vary with the level of fluid in the sheath cubitainer The plenum delivers sheath that has been filtered for air via a bubble filter to the flow cell with minimal flow rate variations Figure A 1 on page 151 shows the fluidics pathway for the BD FACSCanto 150 BD FACSCanto Flow Cytometer Reference M anual Figure A 1 Sheath and sample flow pathways for the BD FACSCanto cytometer Sheath Flow Sample Flow sheath tank y sheath filter interior reservoir plenum bubble filter ng flow cel yoy interr point ator nk You can view the current sheath pressure in BD FACSDiva software by choosing Instrument gt Instrument Status Report In BD FACSCanto clinical software look in the Status window instrument Status Report File Pe nstrument FACSCanto Instrument Status Report Date 2004 02 05 at Parameter X alue Serial Number 1 Loader Status Door Clo Vacuum Status Ok Instrument Info Pump Status Ok Laser Delay Area S Float Status Gk Blue 0 0 1 4CSFlow Pressure 45 Red 0 0 4 ne tae a nindo Evtancion Shutdown Solution Level Ok FACSFlow Pressure 4 50 Cleaning Solution Level Ok STOO hie aio WEIL Laser P
66. hed see Precaution Labels on page Precaution labels A A Use of controls adjustments to the cytometer or performance of procedures other than those specified in the instrument user s guide can result in exposure to hazardous visible laser radiation A A Keep all instrument doors closed during instrument operation W hen XVIII Operated under these conditions the instrument poses no danger of exposure to hazardous laser radiation BD FACSCanto Flow Cytometer Reference M anual General Safety A To prevent injury and maintain data quality do not relocate the cytometer after it has been set up by BD Biosciences service personnel If you need to relocate the cytometer call BD Biosciences Your service representative will arrange the relocation and verify that the cytometer is functioning properly afterwards A M echanical moving parts within the Loader can pinch or injure your hands or fingers To prevent injury by moving parts keep the Loader cover closed while running samples Remove the cover only when installing carousels or performing routine maintenance The Loader will not run with the cover removed Safety and Limitations xix Precaution Labels XX The following precaution labels appear on the BD FACSCanto flow cytometer Loader or fluidics cart to indicate a potential hazard Do not remove these labels Use appropriate precaution to avoid injury by the indicated hazard See the previous sections for mo
67. i ah oe es ee 158 SOINOVER wena ace val sa b 4 4 ae Gb AUS 9 RT BG ba Gg ok OE Ae BS 161 Electronics SY SUC 94 53 aiicoe Ais Nal eaa eg aes ila ea ee en eS 163 PULSES sa Sci Aiea Sea ater corn heady a Ae inci eth inna E Bidet sO Seek Se le ee aed ee 164 Pulse M easurements s 0 4 2 ea too deena aw key be dead beeea ss 165 Appendix B FSC Area Scaling 169 AGONGO FOCA 20 dane dards oad eas ee ene RAS Ana bee RSS 171 Adjusting the FSC Area Scaling Factor cece ee 172 Appendix C Supplies and Replacement Parts 175 Instrument SUD ES saapi song de aan wh arse aaa a aa dardaia a sh opis a Gow sph dosh aed 176 ACCESSORI E oa sisi ar arte ude e oes Biased al ee ewe ae A 176 Other Replacement Parts 0 cece ee eee tees 177 COMI SUIMA DIGS r wlan eg Ge bb 8d ohne GH at a hoe ee ene on 177 MASTU SCRE Dennen baat Gudea each We Maca eau Gone de Sas Adena acy ne Ae 177 Reagents snot Sows eat th ela ees wae Ee te es wa el eels 178 PEON AES ra ee ere wh vats E aaa ety ahi ee Gia eee ee sh aa ees Ge 178 Appendix D Technical Specifications 179 Cytometer Specifications sssaaa Gel eda ake Was Kae eg ee ee 180 EPIVITORIMICMU sais se uiet nrnna Goths M Se e a Boa da det auth Sew Seta 181 Pt AOU Mel ae 525 assert Stel des ates hk ae ese Re ee tee aad eae toe Se bys a arte 181 OPU aeaa gaan aire de ina aes eet aw aa a a e eri 181 FU NGS freer ett owt fa it doh deat a detest ae face aide den eet tey a ental ea 183 SIGMA P FOCESSING inden anaa
68. idics Cart Fuses on page 134 142 BD FACSCanto Flow Cytometer Reference M anual Acquisition Troubleshooting for BD FACSDiva Software Observation No events in plots after clicking Acquire Possible Causes Current Tube pointer not set to current Tube Recommended Solutions Click to move the pointer in front of the appropriate Tube Viewing plots for a different Tube Double click the current Tube in the Browser to display the plots for that Tube Incorrect population s in plot Right click the plot and choose Show Populations Verify that the appropriate populations are displayed Event color matches plot background or set to No Color e Assign a color to the population displayed in the plot e Format the plot to display all events e Verify the population drawing order Current instrument configuration different from optical bench Verify that the current instrument configuration corresponds to the optical bench setup No sample in tube Add sample to tube or install new sample tube Sample not mixed properly M ix sample to suspend cells Sample tube cracked R eplace the sample tube Chapter 5 Troubleshooting 143 Acquisition Troubleshooting for BD FACSDiva Software continued Observation No events in plots after clicking Acquire continued Possible Causes Threshold not set to correct parameter usually FSC Recommended Solut
69. ific You can use both softwares for performing instrument quality control This chapter provides a general overview of the workspace components In BD FACSDiva software and describes instrument controls unique to the BD FACSCanto instrument For an in depth description of software components not described in this chapter refer to the BD FACSD iva Software Reference M anual For information on using BD FACSCanto clinical software refer to the BD FACSCanto Clinical Software U ser s G uide The following topics are covered in this chapter e Workspace Components on page 42 e Instrument M enu Commands on page 43 e Controls in the Instrument Frame on page 48 e Acquisition Controls on page 52 41 Workspace Components When you start BD FACSDiva software the workspace appears Figure 2 1 Frames containing the main application components are displayed within the workspace For an overview of the workspace and to get started using the software refer to the BD FACSD iva Q uick Start G uide In addition to the frames shown in the BD FACSD iva Quick Start Guide the BD FACSCanto also has a Carousel Controls frame that you can access by clicking amp in the Workspace toolbar refer to the BD FACSCanto O ptions Reference M anual for a description of this frame Figure 2 1 BD FACSDiva workspace BD FACSDiva Software File Edit View Experiment Populations Worksheet Instrument Help Gye Oa 0 feeBrowser SH nstrume
70. iguration Set Configuration OK Cancel Before you start any Experiment verify that the instrument configuration contains appropriate parameters for the samples you arerunning and that the instrument optics match the current configuration If needed you can definea custom configuration for your system setup and application A For accurate data results the octagon and trigon arrays must match the current Instrument Configuration In general it is best if you do not switch between configurations too often One way to avoid switching configurations is to design a configuration that includes all the colors your laboratory uses You can assign multiple colors to the same PM T location and laser assignment as shown in the following table Parameter Laser Detector PerCP Cy5 5 Blue B PerCP Blue B PI Blue B Chapter 2 Using BD FACSD iva Software 45 Selections in the Instrument Configuration dialog box determine the parameters listed on the Parameters tab in the Instrument frame When more than one parameter is available for a detector access it through the drop down menu Figure 2 3 Parameters Tab view in Instrument frame Instrument Status Parameters Threshold Compensation Ratio Laser Parameter Log A H w e FITC 500 PE 500 PerCP Cy5 5 Refer to the BD FACSD iva Software Reference M anual for details on how to create your own configuration Instrument Status Report Choose Inst
71. inmi and Analysis FACService Responses gt Introducing TECHNOTES Process Cot Application Notes Previewing BD LSR II Products the BD System Brochures FACSArray A a Product Features Bioanalyzer side menu choice White Papers System Literature Library 168 BD FACSCanto Flow Cytometer Reference M anual Appendix B FSC Area Scaling This appendix provides more information about these topics Adding FSC A on page 171 Adjusting the FSC Area Scaling Factor on page 172 169 The default FSC parameter for the BD FACSCanto flow cytometer is height FSC H If you intend to collect data based on FSC area FSC A you must check the FSC area scaling factor routinely prior to running samples and adjust it as needed You can find more details about the FSC area scaling factor in the BD FACSD iva Software Reference M anual Figure B 1 shows the point at which you should select FSC A as a parameter and check and adjust the FSC area scaling factor Figure B 1 Workflow and adjustment of FSC area scaling factor 1 2 3 4 5 start up run automated a record and shutdown BD FACSCanto setup or assay analyze data clinical software J BD FACSCanto BD FACSDiva BD FACSDiva BD ieee linical software software software software create the Experiment create compensation controls ______s select FSC A and FSC H adjust FSC and SSC ____________ adjust FSC area scaling factor adjust
72. ions Set the threshold to the correct parameter for your application M ultiple Threshold parameters not set correctly Verify the correct Boolean logic And Or was used for the Threshold parameters Threshold channel too low or too high Adjust the Threshold channel Unexpected results after clicking N ext Current Tube pointer on wrong Tube Verify that the pointer is in front of the Tube you want to duplicate before clicking N ext N o fluorescent signal Current instrument configuration different from optical bench Verify the current instrument configuration corresponds to the optical bench setup Wrong filter installed M ake sure the appropriate filter is installed for each fluorochrome Unexpected events in plot Incorrect logic in Population Hierarchy Verify the gating strategy Incorrect population s in plot Right click the plot and choose Show Populations Verify the appropriate populations are displayed Incorrect drawing order Verify the required population is not hidden by another population Right click the plot and choose Order Populations by Count 144 BD FACSCanto Flow Cytometer Reference M anual Acquisition Troubleshooting for BD FACSDiva Software continued Observation Unexpectedly high event rate Possible Causes Threshold channel too low Recommended Solutions Adjust the Threshold channel Sample too concentrated Di
73. khead e To detach the sensor turn the connector counterclockwise and pull sensor aN arr lh en ot 7 A es i e To detach the fluid line press the metal clip on the quick disconnect coupling metal tab Chapter 4 Maintenance 131 3 Unscrew the cap on the cubitainer 4 Removethe cap level sensor sensor and fluid lines and discard into a suitable receptacle 5 Put anew level sensor assembly on the cubitainer or tank H and tighten the cap until it is fully closed M ake sure to use the correct assembly Figure 4 4 on page 133 132 BD FACSCanto Flow Cytometer Reference M anual Figure 4 4 Level sensor assemblies 1 waste level sensor red connector BD FACSFlow level sensor blue connector BD FACS cleaning solution and g BD FACS shutdown solution level sensor ee connector Reattach the sensor line and fluidics line to the cart e To attach the sensor line gently rotate until the connection aligns and then push e To attach the fluid line push the quick disconnect coupling into the port until it clicks into place To ensure that the appropriate solutions are dispensed make sure the label on the container matches the labeled port on the fluidics cart Prime the fluidics lines IMPORTANT Go to Priming on page 101 Chapter 4 Maintenance 133 Replacing the Fluidics Cart Fuses Power surges and other electrical events could cause a fuse to blow Use the following
74. l Adjust the value until the events in the plot are approximately at a 45 degree angle Figure B 2 Correctly adjusted FSC area scaling factor Compensation Controls Unstained Compensation Caontrols Unstained 100 150 700 2450 700 2450 FSC A te 1 000 te 1 000 unadjusted adjusted Appendix B FSC Area Scaling 173 174 BD FACSCanto Flow Cytometer Reference M anual Appendix C Supplies and Replacement Parts This appendix provides a list of supplies and options available for the BD FACSCanto instrument e To order spare parts and consumables from BD Biosciences from within the US call 877 232 8995 or go to www bdbiosciences com In other countries contact your local BD Biosciences representative e To order instrument options contact your sales representative This information is correct at the time of publication for up to date information refer to our website http www bdbiosciences com 175 Instrument Supplies Accessory Kit The instrument is shipped with an accessory kit containing the following items Use these part numbers if you need to order any replacements Sheath filter 1 331394 Cplg Insrt Barb eloow 1 4 Barb 1 4 Flow 2 333072 6 ft cordset for US power 15A 5 15P 320 C 13 337219 2 5 m cordset for Australian power 10A C13 335696 2 5 m cordset for European power 10A C13 335697 2 5 m cordset for UK power 10A C13 R A 335698 Filter bypass assembl
75. l See Replacing the Bal Seal on page 120 Liquid leakage around cytometer base Interior valve failure 1 Turn off the cytometer power 2 Clean up the liquid using proper precautions 3 Call BD Biosciences 140 BD FACSCanto Flow Cytometer Reference M anual Instrument Troubleshooting continued Observation Fluid leaking from SIT or aspirator arm Possible Causes Cytometer still in acquisition mode remove tube sequence not followed correctly Recommended Solutions e For BD FACSDiva software perform the tube removal sequence again step 10 on page 79 even if a tube is no longer on the SIT e For BD FACSCanto clinical software refer to the BD FACSCanto Clinical Software User s G uide Interior valve failure or leak 1 Turn off the cytometer power 2 Clean up the liquid using proper precautions 3 Call BD Biosciences Waste line to fluidics cart disconnected 1 Turn off the cytometer power 2 Clean up the liquid using proper precautions 3 Check that both ends of all waste lines are securely plugged In 4 Turn on the cytometer power 5 If the problem persists call BD Biosciences gt 20 difference between M easured and R eference laser power Flow cell access door open M ake sure the access door is completely closed If this does not resolve the problem contact BD Biosciences BD FACSDiva software not launching C ytometer dat file not found
76. l accessible surfaces with BD FACS cleaning solution A To prevent damage do not use ethanol as a cleaning agent on the exterior of the flow cell or any other instrument surface 3 Weta fresh cloth with DI water and wipe all exposed surfaces to prevent bleach corrosion 4 Wipeall exposed surfaces with a clean dry cloth Decontaminating the Fluidics System for Storage If your cytometer is to be taken out of operation perform the following procedure to clean out the fluidics lines with BD FACS shutdown solution 1 Perform steps 1 through 4 in Decontaminating the Fluidics System Long Clean on page 107 2 Shut down the software and turn off the power to the cytometer Chapter 4 M aintenance 119 Replacing the Bal Seal The Bal seal is a Teflon ring that allows the sample tube to pressurize O ver time this seal becomes worn or cracked and requires replacement R eplace the seal as needed Materials e proper protective equipment e replacement Bal seal Procedure l Turn off the cytometer 2 Ifyou havea Loader follow these steps e Remove the Loader cover e Pull out the drawer e Remove any carousel 120 BD FACSCanto Flow Cytometer Reference M anual e Change the cytometer from automatic to manual mode refer to the BD FACS Loader O ption Reference M anual for instructions If you do not change the cytometer s mode you will not be able to access the Bal seal 3 Movetheaspirator arm to the left and hold i
77. l on page 116 Purging the Bubble Filter on page 117 Cleaning External Surfaces on page 118 D econtaminating the Fluidics System for Storage on page 119 R eplacing the Bal Seal on page 120 Resetting the Cytometer Circuit Breaker on page 124 R econnecting the Fluidics Cart Tubing on page 125 Fluidics Cart Maintenance R econnecting the Fluidics Cart Tubing on page 125 Removing an Air Lock on page 128 Replacing Fluidics Filters on page 111 Replacing the Fluidics Level Sensors on page 130 Replacing the Fluidics Cart Fuses on page 134 114 BD FACSCanto Flow Cytometer Reference M anual Removing Bubbles from the Flow Cell Use the De gas Flow Cell command to remove bubbles from the flow cell 1 Choose Instrument or Cytometer gt Cleaning M odes gt Degas Flow Cell 2 Click OK when the completion message appears De Gassing Flowcell Flowcell successfully de gassed De gas Flow Cell G De gas Flow Cellis complete BD FACSDiva software BD FACSCanto clinical software 3 Check the flow cell for bubbles If you still see bubbles repeat the process Chapter 4 Maintenance 115 Cleaning the Flow Cell Use the Clean Flow Cell command to run a tube of BD FACS cleaning solution through the SIT and flow cell After the procedure is complete the cleaning fluid remains in the SIT and flow cell until you run fluidics startup or shutdown Perform the procedure when poor optical performance indicates that additional cleanin
78. lot of reagent 196 BD FACSCanto Flow Cytometer Reference M anual Lyse No Wash Method Subset Percentages Table E 6 Precision of the BD FACSCanto system with BD FACSCanto software Snes MC Within Run MC Total MCL Within Run MCL Total EPEA yt Precision Precision Precision Precision SD SD SD SD Notes e Testing was performed using commercially available normal and low CD4 control materials BD M ulti Check control M C Jand BD M ulti C heck CD4 low control M CL e The study was run over 20 days at one site and included 40 runs Three devices were used to collect data from control samples stained with one lot of reagent Appendix E Performance Data 197 BD FACSCanto System Linearity For immunophenotyping lymphocyte subsets by flow cytometry the BD FACSCanto instrument with BD FAC SDiva software has been demonstrated to be linear within the following ranges for each fluorescence parameter Table E 7 Table E 7 Linearity Linearity Linearity Measured Measured From To FITC 330 000 99 90 99 90 99 90 M EF FITC PE M EF PE 400 300 000 100 00 100 00 1100 00 PerCP Cy5 5 1 900 1 115 000 99 80 99 90 99 80 MEF Cy5 Fluorophore Unit PE Cy7 RFI 098 100 100 00 100 00 99 60 APC Cy7 025 100 99 80 99 80 99 70 RFI a MEF M anufacturer s Equivalent Fluorescence b RFI Relative Fluorescence Intensity APC 0 025 29 99 70 99 70 99 80 RFI amp APC 198 BD FACSCanto Flow Cytome
79. luorochrome or cell parameter that will be measured at each PM T detector You can add additional parameters to your configuration and choose the appropriate fluorochrome within your software Experiment Detector Arrays On the BD FACSCanto flow cytometer the PM Ts are organized into two basic configurations the octagon and the trigon The octagon has five PM Ts the 158 BD FACSCanto Flow Cytometer Reference M anual trigon has two PM Ts These arrays efficiently direct the emitted light from each fluorochrome to a specific PM T through placement of LP dichroic mirrors and BP filters W hen the collected light leaves the fiber optic cable at the octagon it first meets a 735 LP dichroic mirror Figure A 9 on page 159 The mirror passes through light with wavelengths greater than 735 nm and reflects lower wavelengths on to the next PMT Figure A 9 Light pathway around an octagon 750 810 nm 515 545 nm 564 606 nm 483 493 nm Behind the LP mirror a 780 60 BP filter admits light from 750 nm to 810 nm and substantially blocks other wavelengths The light that finally reaches PM T A will from dyes such as PE Cy7 that emit in this range Appendix A Technical Overview 159 Figure A 10 Excitation and emission spectra of PE Cy7 blue Es bandpass laser light excitation filter d a 550 600 Wavelength nm emission Likewise the 655 LP dichroic mirror for PMT B will reflect light with a wavelength of less than 655 nm
80. lute the sample Sample flow rate too high Decrease the flow rate Air bubble Remove the air bubble Unexpectedly low event rate Threshold channel too high Adjust the Threshold channel Sample not adequately mixed M ix the sample to suspend cells Sample too dilute Concentrate the sample Erratic event rate Sample aggregates Filter the sample Sample contaminated Re stain the sample making sure the tube is clean BD FACSFlow solution cubitainer low Replace the BD FACSFlow solution cubitainer Distorted scatter parameters Instrument settings adjusted incorrectly O ptimize the scatter parameters Air bubble Remove the air bubble Excessive amount of debris in plots Threshold channel too low Increase the Threshold channel Dead cells or debris in sample Examine the sample under a microscope Sample contaminated Re stain the sample making sure the tube is clean Chapter 5 Troubleshooting 145 Acquisition Troubleshooting for BD FACSDiva Software continued Observation High CVs Possible Causes Sample flow rate too high Recommended Solutions Decrease the flow rate Poor sample preparation Repeat sample preparation Old or contaminated quality control QC particles M ake new QC samples and perform the quality control procedure again Window extension too low Increase the window e
81. mand from the Instrument menu In addition fluid level indicators are available in the Instrument frame see Fluid Level Indicators on page 48 Fluidics Startup The Fluidics Startup procedure verifies waste and sheath levels and primes the fluidics system with BD FACSFlow For instructions on using this command see Instrument Startup on page 57 Fluidics Shutdown The Fluidics Shutdown procedure removes sheath from the fluidics system and replaces it with BD FACS shutdown solution For instructions on using this command see Daily Shutdown on page 92 Cleaning Modes BD FACSDiva software contains pre programmed cleaning modes that are activated by choosing the corresponding command from the Instrument gt Cleaning M odes menu Figure 2 2 on page 44 For information on when to use these commands see Scheduled M aintenance on page 96 and Unscheduled M aintenance on page 114 Chapter 2 Using BD FACSD iva Software 43 44 Figure 2 2 Instrument gt Cleaning Modes menu commands Instrument Fluidics Startup Fluidics Shutdown Cleaning Modes Clean Flow Cell Automatic Clean De gas Flow Cell Instrument Configuration Bubble Filter Purge Instrument Name Prime after Tank Refill Instrument Status Report Long Clear Instrument Setup b Standby Automatic Clean Choose Instrument gt Automatic Clean to turn on automatic cleaning A checkmark appears next to the menu command whe
82. n automatic cleaning is in effect Instrument Instrument Fluidics Startup Fluidics Startup Fluidics Shutdown Fluidics Shutdown automatic Cleaning Modes Cleaning Modes automatic cleaning on v automatic Clean Automatic Clean Cleaning off When automatic cleaning is on command checked the system runs Fluidics Startup automatically each time a user logs in or the cytometer resumes running from Standby and runs Fluidics Shutdown each time a user logs out or the cytometer is put into Standby W hen automatic cleaning is off command unchecked you will need to choose the corresponding command to run Fluidics Startup and Fluidics Shutdown Instrument Configuration The BD FACSCanto instrument is equipped with a specific set of lasers filters and dichroic mirrors The Instrument Configuration dialog box lets you define which fluorochromes or cell parameters will be measured at each photomultiplier tube PM T detector BD FACSCanto Flow Cytometer Reference M anual The following default configuration is provided when you install BD FACSDiva software for the BD FACSCanto instrument T he default configuration cannot be edited Instrument Configuration Side Scatter Parameter Current Configuration 2 laser 6 Color Parameter Laser Detector FSC Blue FSC ssc Blue E FITC Blue D PE Blue C PerCP Cy5 5 Blue B PerCP Blue B PE Cy Blue A APC Red B APC Cy Red A Add Conf
83. ndby command is not available when the application is acquiring or recording data running any of the fluidics modes or running a Loader carousel In Standby mode you can set up Experiments or analyze data but you cannot acquire new data all instrument and acquisition controls are disabled Refer to the BD FACSD iva Software Reference M anual for additional information on working offline Chapter 2 Using BD FACSD iva Software 47 Controls in the Instrument Frame To display the Instrument frame click the Instrument button in the Workspace toolbar Along with the controls described in the BD FACSD iva Software Reference M anual the Instrument frame for the BD FACSCanto flow cytometer contains fluid level indicators at the bottom of the frame and reference values on the Laser tab both of which are described in this section Fluid Level Indicators BD FACSDiva software provides fluid level indicators in the Instrument frame Figure 2 5 The FACSFlow and waste Indicators give an approximate Indication of the fluid levels in each tank while the shutdown solution and cleaning solution indicators appear full until the fluid level descends below approximately 20 of the tank capacity When this occurs the corresponding level indicator turns red Figure 2 5 Levels indicators shutdown cleaning solution FACSFlow waste solution empty The system is ready m P B e Green represents the amount of fluid in a tank
84. nt meee Tae _ Lifter Gl a US 8 i color template y Name HS UserName1 E E Folder _001 225 Experiment_001 sS Instr Settings FHB Global Worksheets E B Global Sheet Bi U Tube_oo1 HA Shared View 616104 12 31 3 The system is ready Specimen Keywords Name Collected Global Sheet FE Acquisition Controls xl Tube_001 0 evtis 00 00 00 Next Remove Tube E Acquire E Record Storage Gate All Events v Stopping Gate i All Events v 30000 evt Y Events To Display 1000 evt Medium x Events To Record Flow Rate 42 Bai Worksheet BR SMR E Sef tb Specimen_001 Tube_001 Threshold Rate Threshold Count Electronic Abort Rate Electronic Abort Count Processed Events Elapsed Time 00 00 00 BD FACSCanto Flow Cytometer Reference M anual Instrument Menu Commands M ost BD FACSCanto specific instrument controls are accessed through the Instrument menu Fluidics Startup Fluidics Shutdown Other menu commands Instrument N ame Instrument Cleaning Modes Setup are described in the BD FACSD iva Software Aena cleat R eference M anual Instrument Configuration Instrument Mame Instrument Status Report Instrument Setup p Fluidics Controls T Fluidic control of the BD FACSCanto instrument is completely automated by BD FACSDiva software The software contains pre programmed fluidics protocols that are activated by choosing the corresponding com
85. nt 5A at 115 VAC 2 5A at 230 VAC 500 W 180 BD FACSCanto Flow Cytometer Reference M anual Environment Storage temperature 1 40 C O perating temperature 15 30 C 59 86 F O perating relative humidity 5 80 noncondensing N oise level lt 62 dBA Facilities N o special room requirements Performance Fluorescence threshold sensitivities FITC lt 100 M ESF PE lt 50 M ESF Forward and side scatter sensitivity Sensitivity enables the resolution of platelets from noise Forward scatter sensitivity 1 micron Side scatter sensitivity 0 5 micron Optics Laser Specifications The following Class 3B lasers are mounted on the BD FACSCanto instrument Wavelength Manufacturer Model nm Coherent Sapphire 488 20 488 JDS Uniphase 1144 P 633 Because these lasers are contained within the instrument the BD FACSCanto Isa Class 1 laser product Appendix D Technical Specifications 181 Excitation Optics O ptical platform Beam geometry Emission Optics Collection lens Fluorescence detection Forward scatter detection Side scatter detection Fixed optical assembly Blue and red laser 9 um x 65 um elliptical beam O ptical gel coupled to flow cell N umerical aperture NA 1 2 6 photomultiplier tube detectors Four wavelength ranges detected from 488 nm laser e 750 810 nm PE Cy7 e gt 670 nm PerCP Cy5 5 e 564 606 nm PE e 515 545 nm FITC Two wavelength ranges detected from 6
86. nual 4 Rename the Experiment Immunophenotype 6 Color Expt 8S Instr Settings B Global Worksheets Global Sheet 5 Inthe Experiment Inspector select U se global instrument settings Inspector Experiment Keywords Marne immunophenotype Owner Administrator Modified aea 4 5403 PM rLog Decades for Plots i 4 Log Decades 0 5 Log Decades Use global instrument settings Click 2 to add a Specimen and Tube 7 Expand the Specimen by clicking the 8 Movethe Current Tube pointer to the new Tube E E Immunophenctype Gigg aS Instr Settings H Global Workshee Eb Specimen_OO1 current tube pointer Chapter 3 Running Samples 71 9 Inthelnstrument frame click the Parameters tab Instrument Status Parameters Threshold Compensation Ratio Laser Parameter FSC r SSC FITC PE e ParGP Cys 4 e PE Cy r APC r APC Cy Voltage Log A H 250 OILI oo eee oo eee so eee TEA z Delete ee LL Change add or delete parameters as needed e To change parameters select a parameter and choose a new parameter from the drop down menu e To add a parameter click Add a new line appears select it and choose a parameter from the drop down menu e To delete a parameter select it and click Delete M Tip When thelist contains 6 parameters plus FSC and SSC the Add button becomes inactive Applying the Setup Resul
87. o remove air from the bubble filter Poor CVs might indicate a need to purge the bubble filter Use the Bubble Filter Purge command in BD FACSDiva software to remove air from the bubble filter 1 From the menu choose Instrument gt Cleaning M odes gt Bubble Filter Purge Wait while the purge finishes Chapter 4 Maintenance 117 Progress YD Bubble Filter Purge in progress please wait 2 Click OK when the completion message appears Bubble Filter Purge Bubble Filter Purge is complete 3 Repeat steps 2 through 4 at least four times to remove all air from the filter Cleaning External Surfaces To keep the system free from salt buildup wipe down all external instrument surfaces that have been exposed to sheath fluid A All instrument surfaces that come in contact with biological specimens can transmit potentially fatal disease Use universal precautions when cleaning instrument surfaces Wear suitable protective clothing and gloves A Do not use isopropyl alcohol or ethanol on any cytometer or fluidics cart surfaces They will damage the system Materials e BD FACS cleaning solution DI water e clean lint free cloths or disposable wipes 118 BD FACSCanto Flow Cytometer Reference M anual Procedure 1 Switch off the instrument power and unplug the AC power cord A AA To avoid potential shock always switch off the power and unplug the AC power cord before you begin cleaning 2 Wipeal
88. on tube 9 flow cell i S N O a O O A Olo olO 6 o o tubing adapter lever sample injection ube si tube guide Loader option only 0 Speman aspirator arm bar The following table briefly describes these components where the laser beam intercepts particles tubing that brings sheath and cleaning fluids to and waste away from the flow cell the hollow metal tube that brings sample to the flow cell adapter lever the lever used to change the SIT from manual to automatic loading aspirator arm a movable waste aspiration port aspirator arm bar a metal bar used to push the aspirator arm away from the SIT during manual loading You will install tubes onto the SIT A pump within the fluidics cart pressurizes the interior reservoir which then provides sheath fluid to the flow cell At the same time sample is pushed up the SIT and into the flow cell 28 BD FACSCanto Flow Cytometer Reference M anual When you remove tubes and when the cytometer cleans the SIT between tubes fluid drops will be evacuated into a port on the aspirator arm To activate SIT cleaning push the aspirator arm all the way to the left when you manually unload a tube When you are using the Loader SIT cleaning occurs automatically You do not need to leave a tube of distilled water on the SIT between sample tubes or after daily shutdown Flow Cell
89. ongpass 156 optical theory 155 removing an airlock 128 waste tank cap 104 flow cell access door 27 checking for bubbles 59 cleaning 116 described 28 29 31 problems 138 removing bubbles 115 flow rate adjusting 53 fluidics controls 43 description 150 level indicators 48 58 level sensors replacing 130 priming 101 shutdown 43 92 startup 43 59 system components 27 fluidics cart components 36 controls 38 dimensions 184 fuses replacing 134 level sensor 100 maintenance 98 102 problems 142 reconnecting tubing 125 troubleshooting 140 fluids leaks cytometer 140 leaks fluidics cart 142 leaks SIT 141 levels 48 58 not recommended 118 required 40 fluorescence 154 155 fluorescence signal troubleshooting 144 fluorochromes emission spectra 155 for 488 nm and 633 nm lasers 30 instrument configuration and 44 spectral overlap and 161 Index 209 focusing lens 31 forward scatter FSC described 154 diode 29 frames about 42 acquisition controls 52 See also views windows fuses fluidics cart replacing xv 134 G gating compensation Tubes 80 data 87 general safety xix Iimmunophenotyping analysis 87 Experiment 83 Inspectors troubleshooting 147 installing air filter 108 bypass tubing 129 cubitainers 98 fluidics level sensors 130 fuses 134 tubes 64 instrument configuration 44 controls 43 power 35 QC particles 177 quality control 61 settings optimization 68 speci
90. ons and suggestions for improvement BD FACSDiva software 2004 Becton Dickinson and Company This software is the property of Becton Dickinson and Company Each sale of a stored unit of this software grants the purchaser a nontransferable nonexclusive personal license This software may not be duplicated reproduced or copied in any form or by any means whatsoever except as otherwise permitted by law BD FACSCanto clinical software 2004 Becton Dickinson and Company This software is the property of Becton Dickinson and Company Each sale of a stored unit of this software grants the purchaser a nontransferable nonexclusive personal license This software may not be duplicated reproduced or copied in any form or by any means whatsoever except as otherwise permitted by law BD the BD logo BD Calibrite BD FACS BD FACSCalibur BD FACSCanto BD FACSDiva BD FACSFlow BD Falcon BD M ultiset BD M ultitest and BD Trucount are trademarks of Becton Dickinson and Company JDS Uniphase is a trademark of JDS Uniphase Inc M icrosoft and Windows are registered trademarks of M icrosoft Corporation PE Texas Red is a registered trademark of M olecular Probes Inc Sapphire is a trademark and Coherent is a registered trademark of COHERENT INC Teflon is a registered trademark of E I du Pont de N emours and Company All other company and product names might be trademarks of the respective companies with which they are associated P
91. ous membrane lining the inner surface of the eyelid conjunctiva shedding of the corneal cell layer surface exfoliation and stromal haze T hese symptoms are associated with photokeratitis otherwise known as snow blindness or welder s flash which results from radiant energy induced damage to the outer epidermal cell layer of the cornea T hese effects can be the result of laser exposure lasting only a fraction of a second Safety and Limitations xvii Laser Product Classification Laser hazard levels depend on laser energy content and the wavelengths used A numbered system is used to categorize laser products according to different hazard levels The higher the classification number the greater the potential hazard The BD FACSCanto flow cytometer is a Class laser product per 21 CFR Subchapter J and IEC EN 60825 1 1994 A2 2001 The lasers and the laser energy are fully contained within the instrument structure and call for no special work area safety requirements except during service procedures T hese procedures are to be carried out only by BD Biosciences service personnel Precautions for Safe Operation A A M odification or removal of the optics covers or laser shielding could result in exposure to hazardous laser radiation To prevent irreparable damage to human skin and eyes do not remove the optics covers or laser shielding adjust controls or attempt to service the instrument any place where laser warning labels are attac
92. ower Blue 20 Laser Current Blue 1 57 Laser Power Red 27 2 Event Rate 0 lt ii E BD FACSDiva software BD FACSCanto clinical software Appendix A Technical Overview 151 Test Tube to Flow Cell When you place a test tube on the cytometer sample travels up the sample injection tube SIT in a separate pressurized stream It arrives in the lower chamber of the flow cell at a slight overpressure relative to the sheath fluid The conical shape of the lower chamber creates a laminar sheath flow that entrains and carries the sample particles upward through the center of the flow cell ina stable stream A laser beam then interrogates the sample one cell at a time Figure A 2 The difference in pressure between the sample stream and sheath fluid stream can be used to vary the diameter of the sample stream also known as the sample core Increasing the sample pressure Increases the core diameter and therefore the flow rate Figure A 2 e A higher flow rate is generally used for 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 Figure A 2 Hydrodynamic focusing of the sample core through the flow cell 7s red beam blue beam low sample pressure 10 L min high sample pressure 120 uL min tea eO ea sample sample 152 BD FACSCanto Flow Cytometer Reference M anual
93. patents see the second page of this guide 30 BD FACSCanto Flow Cytometer Reference M anual Fiber optic cables direct the laser light onto beam shaping prisms which in turn transmit the laser light to a focusing lens The lens directs the laser light onto the sample stream within the flow cell Figure 1 4 Figure 1 4 Optical pathway CHO CHO CHo interlock flow cell prisms red He Ne laser focusing lens blue solid state laser When the flow cell access door opens an interlock shutters the laser light and blocks its pathway for safety reasons Chapter 1 Introduction 31 32 Collection Optics From the flow cell laser light is routed to the collection optics which efficiently gather the signals from each laser The BD FACSCanto instrument s collection optics include two detector arrays each consisting of a series of photomultiplier tubes PM Ts arranged in an octagon or trigon Figure 1 5 The octagon containing five PM Ts detects light from the 488 nm blue laser The trigon containing two PM Ts detects light from the 633 nm red laser One PM T in the octagon collects side scatter SSC signals Figure 1 5
94. procedure to replace the fuses Materials e two replacement fuses from the Accessories kit e small screwdriver Removing the Old Fuses A A To protect against shock always turn off the cytometer and unplug the power cord before performing this procedure 1 Shut down the cytometer and turn off the power to the system 2 Unplug the power cord from the wall outlet 3 Unplug the power cord from the fluidics cart Figure 4 5 Fuse door and plug slot for screw driver fuse access door circuit breaker empty socket plug removed Removing the plug allows easier access to the fuse door 134 BD FACSCanto Flow Cytometer Reference M anual 4 Insert a small screwdriver into the slot Figure 4 5 on page 134 and gently ory outward This opens the access door 5 With the screwdriver gently pry the fuse drawer out until you can grip it Power In 6 Remove the fuse drawer 7 Removeand dispose of the old fuses Vv Tip _ It s a good idea to replace both fuses at the same time Installing New Fuses 1 Replace both fuses M ake sure the fuse sits as shown in Figure 4 6 on page 136 Chapter 4 Maintenance 135 Figure 4 6 Proper placement of fuse right wrong A A For protection against risk of fire replace fuses only with those provided by BD Biosciences 2 Makesurethe text for your area s voltage is right side up 3 Slide the drawer back into the inst
95. pt of fitted linear regression line Confidence 1 86 1 52 0 06 0 57 0 62 1 16 0 41 0 31 0 01 0 78 Interval Total number 10 10 108 108 of points used in regression R ange of 40 1 94 1 0 7 69 2 9 4 82 0 0 1 43 9 1 5 44 3 D ata lowest highest value of X R ange of 39 9 93 3 0 5 70 2 9 4 81 3 0 0 41 9 1 9 44 9 D ata lowest highest value of Y 1 0 8 8 Standard error of 35 91 l 0 97 1 12 estimate of the data Correlation 0 993 0 998 0 996 0 994 0 989 coefficient 192 BD FACSCanto Flow Cytometer Reference M anual Notes e Thecomparative method used in regressions was BD FACSCalibur with BD M ultiset software and BD M ultitest IM K Kit with BD Trucount tubes e Themethod used to fit the linear regression for all subsets was least squares e Accuracy studies were conducted over a period of 12 days at two sites Number of replicate determinations used to calculate each mean for X two replicates for CD3 one for all other subsets e Number of replicate determinations used to calculate each mean for Y minimum of two and maximum of four replicates for CD3 and minimum of oneand maximum of two for all other subsets Appendix E PerformanceData 193 Individual Lymphocyte Subset Accuracy Scatter Plots for Lyse No Wash Method Subset Percentages CDS CDS 2 40 60 FACSCalibur 8 CO1B 56 FACS Canto Ce 20 0 20 FACSCalibur
96. r s G uide Cytometer on no response to software commands Bad keyboard or mouse connection Check keyboard or mouse connections to computer Refer to the documentation that came with your workstation Communication failure Ethernet error between computer and instrument To correct the problem follow these steps 1 Turn off the computer and the instrument 2 Reseat the Ethernet cable located next to the power cord on the right side of the flow cytometer 3 Turn on the instrument followed by the computer Chapter 5 Troubleshooting 139 Instrument Troubleshooting continued Observation Cytometer and fluidics cart will not turn on Possible Causes Power cords disconnected from wall socket or cytometer Recommended Solutions Reconnect the power cord to the wall socket or the cytometer Circuit breaker tripped R eset the circuit breaker See Resetting the Cytometer Circuit Breaker on page 124 Sample tube does not fit snugly on SIT Sample tube other than Falcon or BD Trucount brand used Use Falcon brand 12 x 75 mm polystyreneor BD Trucount tubes See System R equirements on page 40 Adapter lever in incorrect position for manual loading M ake sure the lever is in the manual loading position M odify the cytometer appropriately R efer to Changing to M anual Loading in the BD FACSCanto O ptions Reference M anual Bal seal worn Replace the Bal sea
97. r Power Window Extension 7 000 a tFsc Area Scaling 0 54 sp Thesystemisray EBEE The Laser tab also contains Window Extension and FSC Area Scaling controls The Window Extension extends the time in which area is measured The FSC Area Scaling factor adjusts area measurements to be on the same scale as height measurements for signals from the FSC photodiode For standard clinical applications use the default settings Table 2 1 Table 2 1 Default settings Control Default Window Extension 7 000 FSC Area Scaling Varies from instrument to instrument Preset by BD Biosciences BD FACSCanto Flow Cytometer Reference M anual Status Tab This tab lists status messages specific to your Instrument such as communication errors fluidics errors or laser power errors Instrument aa areca when value out of range Tesysensray MBEE Chapter 2 Using BD FACSD iva Software 51 Acquisition Controls The Acquisition Controls frame contains controls used to acquire and record data To display the frame click the Acquisition Controls button on the Workspace toolbar 8 Acquisition controls are visible only when the workstation is connected to the cytometer Along with the controls described in the BD FACSD iva Software R eference M anual the following controls are available for the BD FACSCanto instrument In Figure 2 7 the controls unique to the BD FACSCanto are indicated by a blue outline
98. r coded Chapter 4 M aintenance 125 Figure 4 3 Cytometer and fluidics cart ports System Power Fluidics Cart Connections Power Out Communications Communication Fluid Out cytometer ports fluidics cart ports The ports on the cytometer have their corresponding fluidics cart ports listed in Table 4 2 For example make sure the tubing for f Sheath B connects to the port labeled i Fluid O ut Table 4 3 on page 127 lists port functions Table 4 2 Correspondence of cytometer ports to fluidics cart ports Port or Button on Cytometer Port on Fluidics Cart a System Power b Power Out k Power In c Air In m Air Out d Waste A n Waste e Communications j Communication f Sheath B i Fluid Out g Waste A o Waste h On Off Air In 126 BD FACSCanto Flow Cytometer Reference M anual A AA Do not plug the fluidics cart power cord into a wall outlet Plug the cord into the cytometer only This ensures proper electrical grounding and protects against electrical shock or damage to the instrument Table 4 3 Function of ports buttons and switches Port or Switch a System Power Additional Information Powers both cytometer and fluidics cart b Power Out Connects to fluidics cart c Air In d Waste A Vacuums waste out e Communications Data port f Sheath B BD FACSFlow solution port g Waste A h On Off Auxili
99. re information Label Location s Potential Hazard Waste tank waste tank connectors on fluidics cart waste tank connectors on instrument Risk of exposure to biologically transmissible disease Waste tank Risk of exposure to biologically transmissible disease O N m m m N ear sample injection tube SIT and aspirator arm Risk of exposure to biologically transmissible disease Fluidics cart beneath the power cord Potential of electrical shock if fluidics cart is plugged into the wall outlet Plug into the cytometer only BD FACSCanto Flow Cytometer Reference M anual Location s Potential Hazard On or near all removable Risk of exposure to covers and any place hazardous laser radiation where the laser beam can emerge from the instrument Loader Risk of crushing or CAUTION pinching by moving parts MOVING PARTS Safety and Limitations xxi Label Location s Potential Hazard Loader Risk of crushing or CAUTION i i inching by moving parts Do not run instrument p g by g p with cover removed 334614 B N ear cytometer N one labels BD FACSFlow solution BD FACSFlow solution sheath port sheath port Meaning BD FACSFlow solution sheath Limitations e For In Vitro Diagnostic Use e Notall 12 x 75 mm test tubes and bulk fluids have been qualified for use on the cytometer Use only the following tube
100. relative differences in the size of the cells or particles e SSC indicates relative differences in the internal complexity or granularity of the cells or particles Figure A 4 Forward scatter FSC and side scatter SSC side scatter laser gt forward scatter Fluorescence W hen 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 Some fluorescent compounds emit at a much longer wavelength than their excitation wavelength PerCP absorbs blue light 488 nm and emits red light 675 nm other fluorochromes such as FITC absorb blue 154 BD FACSCanto Flow Cytometer Reference M anual light 488 nm and emit green light 530 nm These differences between excitation and emission allow one laser to excite many fluorochromes The emission spectra for some commonly used fluorochromes are shown in Figure A 5 Figure A 5 Emission spectra of commonly used fluorochromes 100 PESCy7 APC Cy7 nonrelized intensity 0 500 600 wavelength nm Optical Filters O ptical filters modify the spectral distribution of light scatter and fluorescence
101. res method was used to fit the linear regression for all subsets e Accuracy studies were conducted over a period of 12 days at two sites e Number of replicate determinations used to calculate each mean for X two replicates for CD3 one for all other subsets e Number of replicate determinations used to calculate each mean for Y minimum of two and maximum of four replicates for CD3 and minimum of oneand maximum of two for all other subsets 190 BD FACSCanto Flow Cytometer Reference M anual Individual Lymphocyte Subset Accuracy Scatter Plots for Lyse No Wash Method Absolute Counts CDS 4000 FACS Canto cellatuLy 2000 i 2000 g00 FACS Calibur foal lu L CDs 2000 FACS Canto cellatuL 1000 i iad 2000 FACS Calibur cells CDO16 56 1500 1000 FACS Canto cellatuLy 400 i 400 1000 1500 FACS Calibur cells AL CD4 3000 zo00 FACS Canta ells L 1000 i000 Zo00 3000 FACS Calibur calls L cD19 zi FACS Canto cells L iid 1000 zo00 FACS Calibur cellstuly regression confidence c ideal 3 Appendix E Performance Data 191 Lyse No Wash Method Subset Percentages Table E 3 BD FACSCanto system accuracy with BD FACSCanto software Measurement cp3 CD4 CD8 CD19 CD16 56 Unit 1 00 0 99 1 00 0 99 1 00 Slope of fitted linear regression line Confidence 0 98 1 02 0 98 1 00 0 98 1 02 0 97 1 02 0 97 1 02 Interval Interce
102. ription of BD FACSDiva software features specific to the BD FACSCanto cytometer within this manual BD Biosciences recommends that first time user s of this instrument take advantage of operator training offered with the sale of every new instrument The BD FACSCanto Flow Cytometer Reference M anual assumes you havea working knowledge of basic M icrosoft Windows operation If you are not familiar with the Windows operating system refer to the documentation provided with your computer XI Conventions The following tables list conventions used throughout this manual Table 1 lists the symbols that are used in this booklet or on safety labels to alert you to a potential hazard Text and keyboard conventions are shown in Table 2 on page xiii Table 1 Hazard symbols Symbol Meaning CAUTION hazard or unsafe practice that could result in material damage A data loss minor or severe injury or death Ls 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 XII BD FACSCanto Flow Cytometer Reference M anual Table 2 Text and keyboard conventions Convention Use lV Tip Highlights features or hints that can save time and prevent difficulties Italics Italics are used to highlight book titles and new or unfamiliar terms on their first appearance in the text
103. rol Tube in the Browser click Acquire A Do not place any heavy objects on top of the cytometer at any time doing so could cause alteration of data Chapter 3 Running Samples 75 3 Adjust the FSC and SSC voltages to appropriately display the scatter properties of the LWB sample Figure 3 2 Figure 3 2 Voltages adjusted 4 Click the Threshold tab and adjust the FSC Threshold if needed Set the threshold to remove most of the debris without cutting off the lymphocyte population Figure 3 2 5 Adjust the P1 gate on the Unstained Control worksheet to surround only the lymphocyte population Figure 3 2 Select the gate by clicking on the boundary O nce selected you can drag the gate to move it or drag any of the selection handles to change Its size and shape 6 Once the gate is adjusted right click its boundary and choose Apply to All Compensation Controls This applies your gate changes to the P1 gates on the remaining compensation worksheets 7 Select all fluorescence histograms on the Unstained Control worksheet 76 BD FACSCanto Flow Cytometer Reference M anual 8 In the Plot Inspector select the Show Grid checkbox Figure 3 3 For this example do not select Biexponential Figure 3 3 Plot Inspector for fluorescent plots Inspector Plot Title Labels Histograrn Tube bn Controls Unstained Control x Parameter FITC amp Ae Parameter Biexponential X Axis checkbox selected
104. rument Push gently on the drawer until it snaps into place 4 Close the fuse access door 5 Reconnect the power cord to the fluidics cart 6 Plug the cytometer power cord into the wall outlet and switch on the power You have finished replacing the fuses 136 BD FACSCanto Flow Cytometer Reference M anual 5 Troubleshooting Thetipsin this section should help you troubleshoot instrument problems If you require additional assistance contact your local BD Biosciences technical support representative Refer to our website http www bdbiosciences com for up to date contact information For software problems refer to the BD FACSD iva Software Reference M anual or to the BD FACSCanto Clinical Software User s Guide For Loader problems refer to the BD FACSCanto O ptions M anual Troubleshooting suggestions can be found under the following topics e Instrument Troubleshooting on page 138 e Fluidics Cart Troubleshooting on page 142 e Acquisition Troubleshooting for BD FACSDiva Software on page 143 137 Instrument Troubleshooting Observation Possible Causes Flow cell will not fill Fluidics cart power off Recommended Solutions Turn on the power to the fluidics cart by resetting the circuit breaker located on the cart Figure 4 5 on page 134 Always use the power button located on the left side of the cytometer to turn the system off and on No BD FACSFlow or sheath pressure To correct the problem follow
105. rument gt Instrument Status Report to view a report of the current instrument settings The Instrument Status Report is displayed in a separate window with a menu bar above the report header You must be connected to the cytometer to view the Instrument Status report For a full description of the Instrument Status Report refer to the BD FACSD iva Software Reference M anual A BD FACSCanto instrument status report includes the sheath pressure and sample flow rate in the Instrument Info section along with the lasers used corresponding Delay and Area Scaling factors and the Window Extension Figure 2 4 on page 47 46 BD FACSCanto Flow Cytometer Reference M anual Figure 2 4 Supplemental items on a BD FACSCanto Instrument Status Report Instrument Status Report File nstrument FACSCanto erial Humber 1 Instrument Info Instrument Status Report Date 2004 07 23 at 06 23 48 Laser Delay Area Scaling Blue 0 0 1 0 Red 30 0 4 0 Window Extension oO FSC Area Scaling 1 0 FACSFlow Pressure 4 50 Sample Flow Rate Medium Delay is not adjustable and is for information only Standby Choose Instrument gt Standby to disconnect the cytometer from the workstation Laser power is unchanged in Standby mode After a brief pause the workstation disconnects from the cytometer and the menu command changes to Connect If the Automatic Clean command is selected the systems runs Fluidics Shutdown automatically The Sta
106. s 12x 75 mm polystyrene BD Falcon tubes 12 x 75 mm BD Trucount tubes do not use with BD FACSDiva software 12x 75 mm BD FACS 7 color setup bead tubes e When unloading tubes from the SIT always move the aspirator arm all the way to the left to activate SIT cleaning and preserve data integrity e A droplet of approximately 10 50 uL of sheath fluid could remain on the SIT after automatic cleaning Therefore always use a sample size that will not be affected by the addition of this much sheath e Do not place any heavy objects on top of the cytometer at any time doing so could cause alteration of data e For sample and reagent limitations refer to the appropriate reagent package insert xxii BD FACSCanto Flow Cytometer Reference M anual If your instrument is equipped with a Loader be aware that BD Biosciences has not validated Loader mixing for volumes greater than 1 mL BD FACSDiva Software Limitations The BD FACSCanto instrument with BD FACSDiva software was developed for use with the lyse wash method of sample preparation which is not compatible with absolute counting beads BD Biosciences does not recommend using BD Trucount tubes when preparing samples using the lyse wash method Calculation of the lymphocyte subset percentages in BD FACSDiva software involves computing the ratio of reagent positive events to the CD 45 positive lymphocyte events and reporting this ratio as a percentage for each lymphocyt
107. s process when a pulse exceeds the user assigned threshold its height and area are simultaneously calculated by the FPGA Figure A 18 Figure A 18 Pulse measurements thas pulse height Signal intensity 166 BD FACSCanto Flow Cytometer Reference M anual An FPGA calculates pulse height and area in the following manner e Themaximum digitized value of all data points for the pulse becomes the pulse height e Thesum of all data points that occur within a discrete time period becomes the pulse area After height and area calculations occur they are sent to the signal processors Compensation Gating and Scaling The digital signal processors DSPs located on the acquisition boards and on the master board perform several important functions including e correcting for spillover between fluorochromes through a series of mathematical calculations e gating e scaling Scaling The DSPs take the values arrived at by the FPGAS and scale them from 14 to 18 bits When calculating area the electronics add all data points under the pulse in effect increasing the resolution from 16 384 maximum levels of measurement 14 bits to close to 300 000 This is equivalent to approximately 18 bits 262 144 levels For the height to match the area it must be scaled to 18 bits Because data has been converted into 18 bits an 18 bit display is used to keep all data on scale That means a pulse an event will fall into one of 262 144 digi
108. se the tank to malfunction To keep the cap dry place it on the bench label side up when it is not on the tank If you see liquid in the waste cap trap remove the drain plug and fully drain the liquid before you replace the plug For information on laboratory safety refer to the following guidelines NCCLS documents can be ordered online at www nccls org e Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood Body Fluids and Tissue Approved Guideline Wayne PA N ational Committee for Clinical Laboratory Standards 1997 NCCLS document M 29 A e Procedures for the H andling and Processing of Blood Specimens Approved Guideline Wayne PA National Committee for Clinical Laboratory Standards 1990 NCCLS document H 18 A Laser Safety Lasers or laser systems emit intense coherent electromagnetic radiation that has the potential of causing irreparable damage to human skin and eyes The main hazard of laser radiation is direct or indirect exposure of the eye to thermal radiation from the visible and near infrared spectral regions 325 1 400 nm Direct eye contact can cause corneal burns retinal burns or both and possible blindness There are other potentially serious hazards in other spectral regions For cytometers that contain UV lasers excessive ultraviolet exposure produces an intolerance to light photophobia accompanied by redness a tearing discharge from the muc
109. t aspirator arm bar aspirator arm Chapter 4 Maintenance 121 4 Remove the retainer from the SIT by turning it in the direction shown SS fo aD 5 Remove the Bal seal by pulling it off with your fingers Cy A Bal seal a S ooa 6 Install the new Bal seal spring side up 122 BD FACSCanto Flow Cytometer Reference M anual 7 Reinstall the retainer over the SIT and tighten in the direction shown SS a pa 8 Test the installation by manually loading a tube onto the SIT and running fluid 9 Ifyou encounter any problems repeat the procedure Chapter 4 Maintenance 123 t Breaker Ircul Resetting the Cytometer C To reset the circuit breaker for the cytometer follow these steps 1 Toggle the cytometer circuit breaker switch to the on position Locate the circuit breaker switch on the left side of the cytometer main power button O54090 50500 0 0 0 O9202 080895008029080506902 2G260400G0G090620208 26092509050 00000900909 29096250050 H069520202 CKOIG9CK0O0ZOZOKO 0609962 OF 080599292020 o 0595950050 506069020 OZOR O 0207070 O 05069 6590X0 506060020 O22 02000 O 2626962050505 29562020 2506250050 5062620202 OKOH69CKV00ZOZO0KO 06202 oxov OR 09290020 o 0506960050 506069620 OOO 02 p0 0 O 06056060020
110. t abort the process 3 Wait approximately 70 minutes for the cleaning cycle to finish 4 Click OK when the completion message appears Long Cleaning Cytometer successfully cleaned Long Clean i Long Clean is complete i Td am BD FACSDiva software BD FACSCanto clinical software A If the completion message does not appear after 90 minutes verify that there are no error messages in the Status tab of the Instrument frame If the cleaning mode fails see Fluidics Cart Troubleshooting on page 142 Chapter 4 M aintenance 107 5 Choose to shut down or continue e To shut down quit the software and turn off the power to the cytometer e To continue choose Instrument or Cytometer gt Fluidics Startup BD FACS shutdown solution can lyse cells The Fluidics Startup procedure removes BD FACS shutdown solution from the interior reservoir and fluid lines and replaces it with BD FACSFlow solution Replacing the Air Filter The BD FACSCanto instrument has an air filter located in the side door Replace the filter every 6 months to ensure proper instrument performance Materials replacement filter Procedure To change the air filter follow these steps 1 Turn off the power to the cytometer 108 BD FACSCanto Flow Cytometer Reference M anual 2 Open the side door To open the door press the black button A round handle will pop out Twist the handle and pull Chapter 4 Maintenance 109 3 On
111. tal bins or channels when it is eventually assigned to a dot plot or histogram Appendix A Technical Overview 16 7 Embedded Computer The computer embedded within the cytometer communicates with the electronics board and your workstation For more about digital theory refer to Appendix B inthe BD FACSD iva Software Reference M anual For an in depth discussion visit our website at http www bdbiosciences com immunocytometry_systems and download the BD FACSD iva O ption White Paper From the side menu on the Immunocytometry Systems home page choose Products gt Literature gt W hite Papers From the list choose BD FACSD iva O ption White Paper BD Biosciences Clontech Discovery Labware Immunocytometry Systems harmingen BD 3D Biosciences gt Immunocytometry Systems Privacy Terms amp Conditions Latest News Immunocytometry Systems gt CE BD Calib ERA BD FACSCanto Target Value Online Catalog gt BD FACS Bra Inst nt Product Spotlights ia s Sales Reps gt BD Multiwell Support AutoSample Tutorials Customer Education Select One gt Hew BD Flow Cytometry Fastimmune Support Tools Antigen Spe Leukemia and i Application I eee Product Spotlights gt Hew PE Cy7 Customer Support BD p BD Cy Tandem Technical Resources S ee Conjugates ell System for Literature Sorting Antigen gt BD CeflQues System Specific Premier Acc Order Catalog
112. ter Reference M anual For immunophenotyping lymphocyte subsets by flow cytometry the BD FACSCanto instrument with BD FAC SCanto clinical software has been demonstrated to be linear within the following ranges appropriate for each lymphocyte subset using BD M ultitest IM K Kit with BD Trucount tubes Table E 8 Table E 8 Linearity of lymphocyte subsets Linearity Measured Lymphocyte Subset From cells uL Linearity Measured To cells uL Appendix E Performance Data 199 BD FACSCanto System Carryover Carryover of the BD FACSCanto system was determined through evaluation of three instruments Each instrument was tested using the BD FACS Loader automatic sample introduction option as well as manual sample introduction Three high concentration leukocyte samples were consecutively tested followed by three low concentration leukocyte samples Carryover was determined by the following calculation Low 1 Low 3 High 3 Low 3 x 100 Sample introduction method BD FACSCanto system with manual sample introduction 0 027 BD FACSCanto system with use of BD FACS Loader 0 025 200 BD FACSCanto Flow Cytometer Reference M anual Appendix F QC Log This sample quality control QC log can be photocopied or used as a guide in designing your own QC log Figure F 3 on page 205 shows an example of a QC log in use 201 202 BD FACSCanto Flow Cytometer Reference M anual ond i Pas ais ec Se ong Q lt LON
113. the BD Biosciences website www bdbiosciences com b US Patent Nos 4 654 312 4 902 613 5 098 849 Labware 5 mL polystyrene test tubes 12 x 75 mm BD Falcon uncapped 125 per bag capped 125 per bag capped 25 per bag with cell strainer cap 25 per bag Supplier BD Biosciences 877 232 8995 Catalog No 352052 352054 352058 352235 178 BD FACSCanto Flow Cytometer Reference M anual Appendix D Technical Specifications This appendix covers the following topics Cytometer Specifications on page 180 Fluidics Cart Specifications on page 184 179 Cytometer Specifications Dimensions Workspace dimensions Operational clearances cytometer Weight Power requirements Power consumption H eight 63 5 cm 25 In Width 90 2 cm 35 5 in Depth 61 cm 24 in H eight with flow cell access door open 85 cm 33 5 in Unit designed to fit lab bench 55 9 cm 22 in depth Left side 30 cm 11 8 In between unit and other objects or wall to permit proper air flow and access to the main power button and circuit breaker Right side 30 cm 11 8 in between unit and other objects or wall to permit proper air flow Top 22 5 cm 8 9 in between unit and other objects or wall to permit opening of flow cell access door 149 7 kg 330 Ib cytometer only excluding Loader and computer maximum 167 8 kg 370 Ib including Loader 100 115 230 VAC 50 60 Hz Curre
114. thedoor s interior turn the pegs along the upper edge of the filter and remove the old filter 4 Install a new filter and turn the pegs to hold the filter in place Dispose of the old filter It cannot be reused 5 Closethe side door twist the round handle and push it in A To preserve your instrument s best function be careful not to close the door on any tubing or wires 110 BD FACSCanto Flow Cytometer Reference M anual Replacing Fluidics Filters Change the fluid filters as needed when you see increased debris in FSC vs SSC plots or every 6 months Materials e paper towels proper protective equipment e felt tip pen Chapter 4 Maintenance 111 112 Procedure 1 Place a few paper towels beneath the filter to collect drips 2 Remove the filter by pressing the tabs on each quick disconnect coupling metal tab quick disconnect coupling metal tab 3 Position the new filter and connect the couplings M Tip Write today s date on the filter so you will know when to replace it BD FACSCanto Flow Cytometer Reference M anual 4 Open the bleeder valve on top of thefilter and leave it open until fluid seeps out Turn it counterclockwise to open bleeder valve 5 Close the valve Chapter 4 Maintenance 113 Unscheduled Maintenance Perform these maintenance procedures as needed Cytometer Maintenance Removing Bubbles from the Flow Cell on page 115 Cleaning the Flow Cel
115. tion Cleaning Solution ance Cancel BD FACSCanto clinical software BD FACSDiva software Although it is most likely to occur for the sheath filter an air lock can develop in any of the fluidics filters 6 When the prime finishes remove the bypass tubing 7 Reattach the filter to the fluidics cart Chapter 4 Maintenance 129 8 Open the bleeder valve and wait for fluid to seep out close the valve bleeder valve d Continuous Use x Pr essure 60 psi at 20 C 9 Repeat the Prime After Tank Refill Replacing the Fluidics Level Sensors R eplace the fluidics level sensors when instructed to do so by a BD Biosciences service representative Before you replace a sensor try rinsing it with DI water A If you are changing the sensor on the waste tank use proper precaution and wear suitable protective clothing eyewear and gloves All biological specimens and materials coming into contact with them can transmit potentially fatal disease A The waste tank can become pressurized when the cytometer is running Always disconnect the tank from the fluidics cart and wait at least 30 seconds for pressure to dissipate before you remove the level sensor cap Materials e replacement fluidics sensor probe assembly e proper protective equipment 130 BD FACSCanto Flow Cytometer Reference M anual Procedure 1 Ensure that the cytometer is idle not acquiring events 2 Detach the sensor and fluid lines from the cart bul
116. to the SIT The Remove Tube button is disabled during acquisition Flow Rate controls the rate of sample flow through the flow cell Three options are available Low 10 uL min of sample Medium 60 uL min of sample High 120 uL min of sample Flow rates are approximate Chapter 2 Using BD FACSDiva Software 53 54 BD FACSCanto Flow Cytometer Reference M anual 3 Running Samples BD FACSDiva software can work together with the automated setup module in BD FACSCanto clinical software to provide a total package for running samples The following topics are covered in this chapter e Workflow on page 56 e Instrument Startup on page 57 e Instrument Quality Control on page 61 e Data Recording and Analysis on page 83 e Daily Shutdown on page 92 55 Workflow BD Biosciences recommends that when you run samples using BD FACSDiva software you use the automated instrument setup feature in BD FAC SC anto clinical software for instrument QC Then use stained cells to optimize for your assay record and analyze data with BD FACSDiva software Figure 3 1 shows the recommended workflow Figure 3 1 Workflow when running samples in BD FACSDiva software 1 2 3 4 5 record and analyze data BD FACSDiva software optimize for assa BD FACSDiva software shutdown BD FACSDiva start up BD FACSCanto clinical software run automated setu BD FACSCanto linical software
117. ts 1 Right click the Experiment level Instrument Settings HE Experiment_O04 Instr Settings LR Global Sheet Global Worksheets Experiment level Instrument Settings 72 BD FACSCanto Flow Cytometer Reference M anual 2 From the menu choose Apply Setup The Setup Catalog appears Fj Setup Catalog Date 061804 Ik 06 a04 12 42 39 Fh LyseMo Wash 062204 11 26 00 4M LyseiVash 062204 11 26 00 4M 3 Selecta setup from the list BD FACSCanto clinical software generated a Lyse N o Wash and a Lyse Wash setup 4 Click Apply Creating Compensation Controls 1 Choose Instrument gt Instrument Setup gt Create Compensation Controls A dialog appears Confirm Q Experiment s instrument settings already linked to a setup Do you want to remove the link Cancel Chapter 3 Running Samples 73 2 Click OK to unlink Applying a setup imports the clinical software setup Unlinking allows you to optimize the setup The Create Compensation Controls dialog appears Create Compensation Controls Add OK Cancel O ptional Edit the labels associated with parameters as needed Edit labels when your experiment contains samples stained with the same fluorophore conjugated to different antibodies labels that require different compensation values This is especially noticeable in tandem conjugates due to lot to lot variation Refer to the BD FACSD iva Software R eference M anual for
118. ts Run setup again Proceed and optimize current setup with BD FACSCanto clinical software Exit and save current setup BD FACSCanto Flow Cytometer Reference M anual 13 If setup is not successful note the message provided by the software and refer to Setup Troubleshooting in the BD FACSCanto Clinical Software R eference M anual Quitting BD FACSCanto Clinical Software If you are ready to optimize instrument settings using stained samples do the following 1 Choose File gt Exit BD FACSCanto Software A dialog appears 2 Select Exit only Exit BD FACSCanto Clinical Software X 1 Please select how you want to exit Run Fluidics shutdown and exit 8 Exit only 3 ClickOK 4 Launch BD FACSDiva software enter your user name and password and then click OK Chapter 3 Running Samples 67 Optimizing Instrument Settings Before you record data for a sample the PM T voltages compensation and threshold settings should be optimized for each sample type and fluorochrome used T hese adjustments position the cells of interest on scale for scatter and fluorescence parameters This section describes how to perform optimization using the Instrument Setup feature in BD FACSDiva software With this feature you can automatically calculate compensation settings For more information refer to the BD FACSD iva Software Reference M anual If you are performing compensation manually not all steps apply In g
119. ubes 80 data 83 85 RemoveTubebutton 52 79 86 removing tubes 65 replacing fuses xv 134 report instrument status 46 results troubleshooting 144 reusing analyses 90 Index 211 running setup 62 S Safety electrical xv general xix laser xvii sample injection tube SIT described 27 problems 139 140 141 sample optimization about 68 Experiment 70 Samples running 85 saving analyses 90 scaling troubleshooting area 147 scatter parameters distorted 145 scatter light 154 scheduled maintenance 96 seal Bal 120 sensors fluidics 130 setup about 61 age 61 beads 61 failure 66 manual mode 63 running 62 Setup compensation 81 shutting down computer 94 fluidics 43 92 side door cytometer 27 side scatter SSC 154 signals no fluorescent 144 troubleshooting 147 specifications cytometer 180 fluidics cart 184 standby mode 4 7 starting computer 5 7 fluidics 43 59 Instrument 57 supplies instrument 176 T technical assistance xiv threshold troubleshooting 146 threshold adjusting 75 trigon optics 32 34 troubleshooting acquisition 143 CVs 146 electronic aborts 146 event rate 145 Inspector 147 plots 143 144 145 populations 146 scatter parameters 145 signals 144 147 window extension 146 tube removal 52 79 86 Tubes compensation 73 tubes loading 64 removing 65 U unscheduled maintenance 114 user preferences 91 212 BD FACSCanto Flow Cytometer Reference M anual V
120. um 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 164 BD FACSCanto Flow Cytometer Reference M anual Pulse Measurements The pulse of electricity travels from the PM Ts to the electronics boards within the cytometer Figure A 16 Electronics boards PMT PMT PMT PMT PMT PMT SSC FSC diode digital digital signal acquisition signal acquisition processor board processor board master board embedded computer workstation The pulse is amplified and then sent to a 14 bit analog to digital converter ADC on the acquisition board that changes the analog continuous pulse into digital discrete data The ADC does this by sampling the pulse up to 10 million times per second slicing it into 16 384 levels and assigning a measurement to each time sample Figure A 17 on page 166 Appendix A Technical Overview 165 Figure A 17 A digitized pulse measured 10 000 000 times every second digitized into 16 384 levels The ADC sends these numbers to short term memory RAM so that a field programmable gate array FPGA can rebuild a digital version of the pulse for analysis During thi
121. ur Chapter 4 Maintenance 105 2 Loosen the bleeder valve near the top of the filter by turning it counterclockwise M Tip You do not need to completely unscrew the valve if you do it will come off bleeder valve open Continuous Use x Pressure 60 psi at 20 C 3 Wait until fluid seeps out Fluid should seep from the open valve within 30 seconds If no fluid appears make sure the corresponding cubitainer is not empty or detached from the cart If the cubitainer contains fluid and the fluid lines are attached the filter might be airlocked To remove the airlock see Removing an Air Lock on page 128 4 Close the valve by turning it clockwise 5 Repeat steps step 1 through 4 with the next filter 106 BD FACSCanto Flow Cytometer Reference M anual Decontaminating the Fluidics System Long Clean Use the Long Clean command to decontaminate the internal sheath path with BD FACS cleaning solution After decontamination the lines are rinsed with BD FACS shutdown solution The procedure takes 75 minutes to complete Materials e undiluted bleach for waste tank e BD FACS cleaning solution approximately 275 mL e BD FACS shutdown solution approximately 1 100 mL Procedure 1 Ensure fluid levels are adequate empty the waste container if it is full 2 Choose Instrument or Cytometer gt Cleaning M odes gt Long Clean A confirmation dialog appears Click OK to continue Once you have begun the Long Clean you canno
122. user s guides the protection provided by the equipment might be impaired K eep this safety information available for reference Electrical Safety A A Lethal electrical hazards are present in some lasers particularly in laser power supplies M any portions of the electrical system including the printed circuit boards are at a dangerous voltage level To prevent shock injury or damage to the instrument follow these guidelines Turn off the power switch and unplug the power cord before servicing the instrument unless otherwise noted e Connect the equipment only to an approved power source Do not use extension cords H ave an electrician immediately replace any damaged cords plugs or cables e Do not remove the grounding prong from the power plug H ave a qualified electrician replace any ungrounded receptacles with properly grounded receptacles in accordance with the local electrical code A A Protect against the risk of fire by replacing fuses only with those of the specified type and rating XV A AN Do not plug the fluidics cart power cord into a wall outlet Plug the cord into the cytometer only This ensures proper electrical grounding and protects against electrical shock or damage to the instrument Biological Safety A Aal biological specimens and materials coming into contact with them can transmit potentially fatal disease To prevent exposure to biohazardous agents follow these guidelines e Handl
123. w cytometer power panel main power button 0626202 0626202 o o O O O o o o o 26 O0 o o O o o O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O cytometer circuit breaker The instrument circuit breaker is located next to the power cord Figure 1 6 The breaker will need to be reset if there is a power surge in the laboratory Chapter 1 Introduction 35 36 Fluidics Cart A separate self contained fluidics cart provides filtered sheath and cleaning fluids to and collects waste from the instrument Figure 1 7 The 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 Figure 1 7 Fluidics cart cubitainer 10 L waste container pressure gauge door access knob filters BD FACSCanto Flow Cytometer Reference M anual Containers and Ports The fluidics cart holds one 10 L waste container one 20 L BD FACSFlow cubitainer and two 5 L auxiliary cleaning fluid containers Figure 1 8 Use the waste container provided with the system do not substitute other containers Figure 1 8 Fluidics cart containers and ports BD FACS shutdown BD FACS cleaning solution solution port and cubitainer port and cubitainer lt BD
124. ware BD FACSCanto clinical software 3 Wait while the instrument primes the specified lines Chapter 4 Maintenance 101 4 Click OK when the completion message appears Priming Priming complete Tank Prime Status G Tank prime is complete BD FACSDiva software BD FACSCanto clinical software Emptying the Waste Container A 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 precaution and wear suitable protective clothing eyewear and gloves waste port gt disposable waste cap trap waste tank 1 Ensure that the cytometer is not acquiring events 102 BD FACSCanto Flow Cytometer Reference M anual 2 Detach the waste container s sensor and fluid line from their respective ports on the cart e To detach the sensor turn the connector counterclockwise and pull e To detach the fluid line press the metal clip on the quick disconnect coupling censor KF aK G amp G AN Waste A metal clip A The waste tank can become pressurized when the cytometer is running Always disconnect the tank from the fluidics cart before you empty it Wait at least 30 seconds for pressure to dissipate before you remove the waste
125. without holding the tube the tube could fall off the SIT and expose you to potentially blohazardous sample e Remove the tube from the SIT e Release the aspirator arm SIT cleaning occurs when the aspirator arm comes to center e When the Progress dialog disappears you can load the next tube onto the SIT Do not changethe PM T voltages after the first compensation Tube has been recorded To calculate compensation all Tubes must be recorded with the Same PM T voltage settings If you need to adjust the PMT voltage for a subsequent compensation Tube you will need to record all compensation Tubes again Chapter 3 RunningSamples 79 Calculating Compensation Before you calculate compensation you need to record data for each single stained control 1 Install the first stained control tube onto the cytometer M ake sure the Remove tube dialog disappears first 2 IntheAcquisition Controls frame click the N ext button N ext moves the Current Tube pointer to the next Tube in the Browser A Do not place any heavy objects on top of the cytometer at any time doing so could cause alteration of data Click Acquire Verify the P1 gate still encircles the population of interest To record data click Record When recording is finished click Remove Tube Install the next tube onto the SIT o nr oo ue A W Repeat steps 2 through 7 until data for all stained control tubes has been recorded Adjusting Gates N ow that data h
126. xtension H igh electronic abort rate gt 10 of system event rate Event rate too high Decrease the event rate Sample aggregated Filter the sample Sample too concentrated Dilute the sample Threshold channel too low Increase the threshold channel Window extension too high Decrease the window extension Fewer events than expected in gated population Events left out of gate When drawing a gate make sure events on the axis are included Plot zoomed Unzoom the plot or make the gate bigger Window extension set incorrectly Adjust the window extension Increasing threshold results in decreased Area signal Window extension too low Slightly increase the window extension to maximize Area signal NOTICE Increasing the window extension too much results in more electronic aborts or high CVs 146 BD FACSCanto Flow Cytometer Reference M anual Acquisition Troubleshooting for BD FACSDiva Software continued Observation Area measurement off scale while H eight measurement on scale for FSC Possible Causes FSC Area Scaling too high Recommended Solutions D ecrease area scaling to move the Area measurement on scale If necessary adjust area scaling to make the Area measurement match the H eight measurement No signal in FSC A or all events against left axis in FSC A FSC Area Scaling set to 0 Set FSC Area Scaling correctly See
127. y 1 335760 Bal seal for SIT 6 343509 Blank 3 1 2 in disk 1 343572 12 x 75 mm test tubes bag of 125 343675 10 L waste tank 1 333503 Vented cap for waste tank 1 336482 Waste cap label 1 336326 5 0 A 250V Slo blo fuse 2 90069 22 176 BD FACSCanto Flow Cytometer Reference M anual 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 replacement parts Sheath sensor probe 5 level 334914 Waste sensor probe 6 level 334915 Auxiliary sensor probe 1 level 334911 Air filter side door 336303 Fuses fluidics cart 96 20054 00 Bypass tubing 336768 Bal seal retainer Consumables 335513 Instrument Setup Particle BD FACS 7 color setup beads Supplier BD Biosciences 877 232 8995 Catalog No 335775 Appendix C Supplies and Replacement Parts 177 Reagents Reagent BD FACSFlow sheath fluid Supplier BD Biosciences 877 232 8995 Catalog No 340398 US and Latin America 342003 other countries BD FACS cleaning solution BD Biosciences 340345 BD FACS shutdown solution BD Biosciences 334224 M onoclonal antibodies BD Biosciences a BD FACS lysing solution BD Biosciences 349202 a Refer to the BD Biosciences mmunocytometry Products Catalog or

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