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BS LSRFortessa Cell Analyzer User's Guide

1. 68 BD LSRFortessa Cell Analyzer User s Guide The CST Mismatch dialog appears CST Mismatch The settings From CST are different From those on the cybometer A Do you want to use the CST values c Dont show this message again for current login session Remember ty decision Use CST Settings Keen Current Settings Click the Details button to verify which cytometer settings will be updated 13 Click Use CST Settings By selecting Use CST Settings the laser delay area scaling and other cytometer settings will be updated to the latest settings from the performance check Next step Continue the optimization of your cytometer for an experiment or sample type as described in Setting up an experiment page 69 Chapter 5 Optimizing cytometer settings 69 Setting up an experiment About this topic Creating an experiment This topic describes how to create an experiment in a new folder specify the parameters of the experiment and add compensation tubes To create an experiment 1 Click the buttons on the Workspace toolbar to display the following windows as needed Browser Cytometer Inspector Worksheet Acquisition Dashboard When you add elements or make selections in the Browser the Inspector displays details properties and options that correspond to your selection Click the New Folder button on the Browser toolbar to add a new folder Click the
2. because compensation subtracts a percentage of the fluorescence intensity The following figure illustrates this principle Although the signals differ in intensity the percentage of the FITC spillover into the PE detector remains constant 110 BD LSRFortessa Cell Analyzer User s Guide FITC PE Different intensity FITC signals Same proportion or percentage of spectral overlap in PE channel Normalized intensity 500 nm S50 Arm 600 nm 650 nm 700 nm Wavelength nm Chapter 7 Technical overview 111 About electronics About this topic This topic describes the electronics in the BD LSRFortessa flow cytometer Pulse As cells or other particles pass through a focused laser beam they scatter the laser light and can emit fluorescence Because the laser beam is focused on a small spot and particles move rapidly through the flow cell the scatter or fluorescence emission signal has a very brief duration only a few microseconds This brief flash of light is converted into an electrical signal by the detectors The electrical signal is called a pulse The following figure illustrates the anatomy of a pulse 1 A pulse begins when a particle enters the laser beam At this point both the beam intensity and signal intensity are low Signal intensity Time 2 The pulse reaches a maximum intensity or height when the particle reaches the middle of the beam where the beam and signal intensity are the brightest The peak
3. BD LSRFortessa Cell Analyzer User s Guide container cap assembly becomes clogged air cannot be vented from the container causing it to swell under pressure If you observe swelling of the waste container loosen the cap to relieve the pressure and immediately replace the air filter See Replacing the waste air filter page 49 for instructions Removing the air filter or the waste container cap assembly without first relieving the pressure might generate an unnoticeable aerosol Use appropriate precautions when troubleshooting a clogged air filter Wear suitable protective clothing eyewear and gloves i Caution If the air vent filter on the top of the waste Procedure To prepare the waste container 1 Verify that the flow cytometer is in standby mode Press the STNDBY button on the control panel if necessary 2 Disconnect the orange waste tubing and the black level sensor line from the waste container Keep the lid on the waste container until you are ready to empty it 3 Empty the waste container Caution The waste container is heavy when full When emptying it use good body mechanics to prevent injury 4 Add approximately 1 L of bleach to the waste container and close it 5 Reconnect the orange waste tubing and make sure it is not kinked 6 Reconnect the level sensor line Chapter 3 Cytometer setup 33 Priming the fluidics About this topic This topic describes how to prime the fluidics system
4. Internet access is not required to use the Help system 11 Supplies and consumables This chapter covers the following topics Ordering information page 176 Particles page 177 Reagents page 178 Equipment page 179 176 BD LSRFortessa Cell Analyzer User s Guide Ordering information To order spare parts and consumables from BD Biosciences e Within the US call 877 232 8995 e Outside the US contact your local BD Biosciences customer support representative Worldwide contact information can be found at bdbiosciences com Chapter 11 Supplies and consumables 177 Particles About this topic This topic lists the QC and CS amp T particles available QC particles Particle Laser Supplier Catalog No SPHERO Rainbow All BD Biosciences 559123 Calibration Particles 8 peaks SPHERO Ultra Rainbow All Spherotech Inc URFP 30 2 Fluorescent Particles single peak DNA QC Particles kit Blue 488 nm BD Biosciences 349523 CS amp T particles Particle Laser Supplier Catalog No BD Cytometer Setup and e UV 355 nm and 375 nm BD Biosciences 641319 Tracking CS amp T beads Violet 405 nm 1 vial e Blue 488 nm an e Red 640 nm e Yellow green 561 nm e Green 532 nm 178 BD LSRFortessa Cell Analyzer User s Guide Reagents Reagent Supplier Catalog No BD FACSFlow sheath fluid BD Biosciences 342003 BD FACS sheath solution with BD Biosciences 336524 surfactant recommended for use with the HTS
5. intensity or height of the pulse is measured at this point Signal intensity Time 112 BD LSRFortessa Cell Analyzer User s Guide 3 As the particle leaves the beam the pulse trails off below the threshold gt Q z a z 5 C N ime Pulse measurements The pulse processors measure pulses by three characteristics height area and width Pulse Height oe D S 9 a Pulse Area Baseline Threshold 0 volts I I I I l O e i Window Gate Window Extension gt l 11 1 J Mindow Extension y Window Gate Full Window Extension e Height The maximum digitized intensity measured for the pulse e Area The integration of all the digitized samples over time where time is the window gate plus 1 2 the window extension added before the initial threshold plus the other half of the window extension value added after the pulse drops below threshold e Width Calculates A _ x 64 000 Height Digital electronics Threshold Chapter 7 Technical overview 113 BD LSRFortessa electronics digitize the signal intensity produced by a detector The digitized data is stored in memory and further processed by the electronics to calculate e Pulse height area and width e Compensation e Parameter ratios These results are transferred to your workstation computer for further processing by BD FACSDiva software For more information about digital theory see Digital Theo
6. 3 About the BD LSRFortessa documentation 0 0 0 0 cece eee eee eee 5 Technical Assistance ovate taba betwee des CAS ode Bees Heke bed ek eos 7 Chapter 2 Introduction 9 lastrum Ove EW 612 aeneo neee ERE EE EEE RA 10 COMPONENTS asst orice ested a a a E aa a aa Gch a maa te AN a E T 11 Pde 5 5 46k auna eaaa r r a aa a aaa T aT ETS 13 Sheath and waste containers lt 6 440004 haa a e T E ESA 17 ICS arse Shas oho ee tae ot al Soe Ne dele Ao Genie ana saan ome le Aes ahs oh len 18 WOCkStatiOn gae nails Baek Fee RS aE ONES ON ORR ERRORS 21 Chapter 3 Cytometer setup 23 Starting the cytometer and computer 0 eee eee eee eens 24 Preparing the sheath container 5 6 e 40 i 0 0 5 stew aden Area BL a we eel ee 26 Removing alt buUDDICS ee rinner tiraa ee ee wee eid eee A BE es 28 Preparing the Waste COnN E 2 5 sso 6b SUN Saw ee a Me we he we Se ee 30 Primine te fl G1 diCS acesccacees haar hacase ay e Ree a gk ee ahd do alae ara 33 About the optical filters and mirrors 0 ee eee eens 34 Changing optical filters and mirrors 0 ccc eee eens 36 Custom configurations and baselines 0 cece eee eee eens 37 vi BD LSRFortessa Cell Analyzer User s Guide Chapter 4 Maintenance 39 Maintenance OVERVICW purdan 64 ch eed ota ee ek Nees Sees 40 Cleaiine the mhOies exit aad 8 ee 8 Ghat ees Saw ee Aaa ee ee 41 Shutting down the cytometer 0 2 ce eee tent eens 43 Flushing The systern 4 04th Ae i Paes ORR aS eA O
7. 500 nm Shortpass 480 520 460 500 540 Transmission 450 500 550 Wavelength nm AS 106 BD LSRFortessa Cell Analyzer User s Guide Bandpass BP filters 100 Transmission A BP filter transmits a relatively narrow range or band of light BP filters are typically designated by two numbers The first number indicates the center wavelength and the second refers to the width of the band of light that is passed For example a 500 50 BP filter transmits light that is centered at 500 nm and has a total bandwidth of 50 nm Therefore this filter transmits light between 475 and 525 nm Bandpass 460 500 540 480 520 450 500 550 Wavelength nm The performance of an optical BP filter depends on the optical transmission Sample transmission curves are shown in the following figure A filter with a narrower steeper transmission curve generally yields higher performance The transmission specifications depend on the construction of the filter Higher performance filters generally have multiple layers of optical coatings and unique manufacturing processes For more demanding multicolor applications higher performance filters are available through the Special Order Research Program Contact your local BD Biosciences sales representative for details Dichroic mirrors Chapter 7 Technical overview 107 100 50 Transmission n m m m Fal 450 500 550 Wavelength nm Dic
8. Analyzing data page 88 Reusing an analysis page 94 82 BD LSRFortessa Cell Analyzer User s Guide Data recording and analysis workflow About this topic About the examples This topic outlines the basic acquisition and analysis tasks using BD FACSDiva software The examples in this chapter is from two 4 color bead samples with the following fluorochromes e FITC e PE e PerCP CyS 5 e APC If you use a different sample type or if you have skipped the optimization steps in Optimizing cytometer settings page 57 your software window content names of folders and experiments and your data plots and statistics might differ from those shown here You might also need to modify some of the instructions in the procedure For additional details on completing some of the following steps see the BD FACSDiva Software Reference Manual This procedure builds on the results obtained in Optimizing cytometer settings page 57 Workflow for recording and analyzing data Chapter 6 Recording and analyzing data 83 Recording and analyzing data consists of the following steps Step Description 1 2 3 Preparing the workspace page 83 Recording data page 85 Analyzing data page 88 Reusing an analysis page 94 Preparing the workspace About this topic Procedure This topic describes how to prepare the workspace and apply application settings to your experiment before recording data To prepare the
9. When to prime the Sometimes air bubbles and debris may become lodged in the fluidics flow cell This is indicated by excessive noise in the forward and side scatter parameters In these cases it is necessary to prime the fluidics system Procedure To prime the fluidics 1 Move the tube support arm to the side 2 Remove the tube from the SIP 3 Press the PRIME fluid control button to force the fluid out of the flow cell and into the waste container Once drained the flow cell automatically fills with sheath fluid at a controlled rate to prevent bubble formation or entrapment The STNDBY button turns amber after completion 4 Repeat the priming procedure if necessary 5 Install a 12 x 75 mm tube with 1 mL of DI water on the SIP and place the support arm under the tube Leave the cytometer in standby mode Related topics e Cytometer troubleshooting page 116 34 BD LSRFortessa Cell Analyzer User s Guide About the optical filters and mirrors About this topic Filter and mirror configurations This topic provides a description of the optical filters and mirrors Each PMT except the last PMT in every detector array has two slots in front of it a filter slot and a mirror slot Filter slot Mirror slot e Filter slot The slot closer to the PMT that holds a bandpass filter holder e Mirror slot The slot farther from the PMT that holds a longpass dichroic mirror holder The filters steer progressi
10. acridine orange application settings applying 84 creating 73 area scaling 168 arm tube support 15 aspiration sample rapid 117 assistance technical 7 Index Bal seal ordering 179 replacing 52 bandpass BP filters detector array placement 20 holder 34 theory 106 base configurations 138 battery changing 47 test 46 BD FACSClean solution 178 BD FACSDiva software See software BD FACSFlow sheath fluid 178 BD FACSFlow solution 27 BD FACSFlow supply system 24 43 BD FACSRinse solution 42 BD Falcon tubes 179 BD High Throughput Sampler HTS 16 blank optical holders 35 bleach 40 178 blue 488 nm laser 139 141 149 154 BP See bandpass filters bubbles removing air 28 buttons fluid control 14 sample flow rate control 13 182 BD LSRFortessa Cell Analyzer User s Guide C calculating compensation 80 capacity waste container 17 cleaning solutions 40 compensation calculating 80 control tubes 76 recording settings 77 theory 108 components cytometer shown 11 sheath container shown 26 sheath filter shown 50 waste container shown 31 computer system about 21 configuration base 133 BD FACSDiva_ 61 maps 132 containers sheath 17 26 waste 17 30 32 control panel cytometer 12 controls fluidics 12 single stained 60 82 conventions keyboard 4 safety symbols 3 text 4 creating analysis objects 88 global worksheets 83 statistics view 88 CS amp T particles 177 cuvette flow cell 164 CV troubleshooting 125 cytome
11. battery You need the following supplies to change the battery e Small flat head screwdriver e A 9 volt battery To change the battery 1 Insert the tip of a flat head screwdriver into the slot and gently slide the battery drawer out 2 Remove the drawer 48 BD LSRFortessa Cell Analyzer User s Guide 3 Remove the battery from the drawer 4 Place a new 9 volt battery into the drawer The markings in the battery drawer show the correct battery orientation 5 Slide the drawer into the bracket until you feel a click 6 Test the new battery Chapter 4 Maintenance 49 Replacing the waste air filter About this topic Procedure A This topic describes the procedure for replacing the waste air filter An air filter is located on the cap assembly of the waste container Caution Biohazard Treat a contaminated air filter as biohazardous waste To replace the air filter 1 Remove the air filter Hold the silicone tubing with one hand and pull off the filter with the other hand 2 Insert a new air filter into the silicone tubing 3 Verify that the cap assembly on the container is tightened 50 BD LSRFortessa Cell Analyzer User s Guide Changing the sheath filter About this topic This topic describes how to change the sheath filter The sheath filter is connected in line with the sheath line It filters the sheath fluid as it comes from the sheath container When to change the We reco
12. designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his or her own expense Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits This Class A digital apparatus meets all requirements of the Canadian Interference Causing Equipment Regulations Cet appareil num rique de la classe A respecte toutes les exigences du R glement sur le mat riel brouilleur du Canada Compliance information NOTICE This laboratory equipment has been tested and found to comply with the EMC and the Low Voltage Directives This includes FCC Part 15 compliance for a Class A Digital Device CAUTION Any unauthorized modifications to this laboratory equipment may affect the Regulatory Compliance items stated above History Revision Date Change made 647247 Rev A 5 2009 New document 23 11093 00 Rev A 3 2010 Updated Contents Chapter 1 About this guide 1 Wat iS Ud COVERS aa bsvics aaa Sood Goa R eark ae Oe Nha ENEE 2 Conventions used in this guide se echaie va enka S584 Cho Rao we SKA
13. features of BD FACSDiva software Before you record data for a sample optimize the cytometer settings for the sample type and fluorochromes used Compensation setup automatically calculates compensation settings If you choose to perform compensation manually not all of the following instructions apply For detailed instructions see the BD FACSDiva Software Reference Manual If you are performing the procedures in this workflow for the first time you should be familiar with BD FACSDiva software concepts workspace components cytometer and acquisition controls and tools for data analysis To become familiar with BD FACSDiva software perform the tutorial exercises in Getting Started with BD FACSDiva Software For additional details see the BD FACSDiva Software Reference Manual Start the BD LSRFortessa cytometer and perform the setup and QC procedures See Cytometer setup page 23 Chapter 5 Optimizing cytometer settings 59 Workflow for optimizing Cytometer optimization consists of the following steps settings Step Description 1 Verifying the configuration and user preferences page 61 2 Running a performance check page 64 3 Setting up an experiment page 69 4 Creating application settings page 73 5 Recording compensation controls page 76 6 Calculating compensation page 80 Note Application settings are optional and do not have to be saved for the experiments However they are useful for optimiz
14. its maximum capacity 8 L When an overfull tank is pressurized erratic cytometer performance can result The waste container has a capacity of 10 L An alarm sounds when the container becomes full e Preparing the sheath container page 26 e Preparing the waste container page 30 18 BD LSRFortessa Cell Analyzer User s Guide Optics About this topic This topic describes the optical components for the BD LSRFortessa cytometer including e Detector arrays e Laser options e Optical filters e Signal detectors Detector arrays The BD LSRFortessa detector arrays consist of e Octagons Array of PMTs and filters that can detect up to eight signals e Trigons Array of PMTs and filters that can detect up to three signals Locked cover lasers accessible by BD Service only g pp LSRFortess Front door panels trigons Side drawer octagons or trigons Laser options Optical filters Chapter 2 Introduction 19 The BD LSRFortessa flow cytometer can be configured with up to four lasers listed in the following table The cytometer can also be upgraded with lasers from this list or configured with up to five lasers from a variety of wavelengths through our Special Order Research Program Laser Wavelength nm Power mW Blue 488 50 Red 640 40 Violet 405 50 UV 355 20 Optical filters attenuate light or help direct it to the appropriate detectors The name and spectral characteristics of each filter appear
15. lip of the sheath lid is seated correctly and has not slipped out of position If the gasket is not seated correctly the tank will not pressurize properly Close the sheath lid and tighten the clamp knob to finger tight Ensure that the sheath fluid line blue is not kinked 28 BD LSRFortessa Cell Analyzer User s Guide Related topics e Removing air bubbles page 28 e Changing the sheath filter page 50 Removing air bubbles About this topic This topic describes how to remove trapped air bubbles in the sheath filter and the sheath line Air bubbles can occasionally dislodge and pass through the flow cell resulting in inaccurate data Procedure To remove air bubbles 1 Check the sheath filter for trapped air bubbles Cytometer fluid line roller clamp not visible Vent fitting Vent line Chapter 3 Cytometer setup 29 If bubbles are visible gently tap the filter body with your fingers to dislodge the bubbles and force them to the top Note When removing air bubbles do not vigorously shake bend or rattle the sheath filter or you might damage it Direct the vent line into a beaker and press the small button at the end of the vent fitting against the side of the beaker until a steady stream of fluid empties from the filter Press small button Vent fitting Tilt the filter and verify that no trapped air remains in the filter Repeat steps 3 and 4 until no air is observed in the filter C
16. moving through a laser beam at a given time The difference in pressure between the sample stream and sheath fluid stream can be used to vary the diameter of the sample core Increasing the sample pressure increases the sample flow rate thereby increasing the core diameter The flow rate should be set according to the type of application you are running e A higher flow rate is generally used for qualitative measurements such as immunophenotyping The data is less resolved but is acquired more quickly e A lower flow rate is generally used in applications where greater resolution and quantitative measurements are critical such as DNA analysis Proper operation of fluidic components is critical for particles to intercept the laser beam properly Always ensure that the fluidics system is free of air bubbles and debris and is properly pressurized 100 BD LSRFortessa Cell Analyzer User s Guide About optics About this topic Optics system Light scatter This topic describes the optics system and provides information about e Light scatter on this page e Fluorescence page 101 e Optical filter theory page 103 e Compensation theory page 108 The optics system consists of lasers optical filters and detectors Lasers illuminate the cells or particles in the sample and optical filters direct the resulting light scatter and fluorescence signals to the appropriate detectors When a cell or particle passes through a foc
17. on page 33 Set the sample flow rate lower Ensure that the sheath container lid is tight and all connectors are secure Flush the system See Flushing the system on page 44 Remove the dichroic mirrors reverse them and replace them in the optical slot Replace the waste vent filter Caution Pressurized contents might spray Use appropriate cautionary measures 1 Slowly loosen the waste tank cap to relieve the pressure in the waste tank 2 Remove the waste vent filter by rotating counter clockwise 3 Install a new waste vent filter Repeat sample preparation 126 BD LSRFortessa Cell Analyzer User s Guide Possible causes Sample not diluted in same fluid as sheath fluid Optical filters are incorrect Old or contaminated QC particles Instrument is not warmed up Laser not functioning Optical alignment problem Optical filters are incorrect Recommended solutions Dilute the sample in the same fluid as you are using for sheath Check the configuration and insert the correct filters Make new QC samples and perform the quality control procedure again Wait 30 minutes for the instrument to warm up Contact BD Biosciences Contact BD Biosciences Check the configuration and insert the correct filters Electronics troubleshooting About this topic Cytometer Disconnected in cytometer window Chapter 8 Troubleshooting 127 This topic describes possible probl
18. option Monoclonal antibodies BD Biosciences See the BD Biosciences Product Catalog or the BD Biosciences website bdbiosciences com BD FACS lysing solution BD Biosciences 349202 BD FACSRinse solution BD Biosciences 340346 BD FACSClean solution BD Biosciences 340345 Dyes and fluorochromes BD Biosciences Molecular Probes or Sigma Chlorine bleach Clorox or other major 5 sodium hypochlorite supplier to ensure that the bleach is at the correct concentration and free of particulate matter Chapter 11 Supplies and consumables 179 Equipment Equipment item Supplier Catalog No Bal seal BD Biosciences 343509 O ring sample tube 343615 Sheath filter assembly 345743 BD Falcon polystyrene 352052 test tubes 12 x 75 mm 352054 352058 Need more information fast Information about this product is also available in your software s Help system You can keep the Help window open while you use the software or print the information directly from the window Internet access is not required to use the Help system Numerics 355 nm UV laser 405 nm violet laser 488 nm blue laser 532 nm green laser 153 162 561 nm yellow green laser 152 161 640 nm red laser 142 144 150 156 148 152 159 145 147 151 157 139 141 149 154 A acridine orange AO 41 air bubbles removing 28 alarm battery test 46 waste container 17 analysis data 82 immunophenotyping 88 reusing 94 saving 94 antibodies 101 AO See
19. population PE positive in the population hierarchy Inthe FITC vs PerCP Cy5 5 plot draw a gate around the PerCP Cy5 5 positive population Name the population PerCP Cy5 5 positive in the population hierarchy Inthe FITC vs APC plot draw a gate around the APC positive population Name the population APC positive in the population hierarchy 10 Format the statistics view a Right click the statistics view and select Edit Statistics View b Click the Header tab and select the Specimen Name and Tube Name checkboxes c Click the Populations tab and select all populations except All Events Clear the Parent Total and Events checkboxes d Click the Statistics tab and select the mean for each of the fluorescence parameters 92 BDLSRFortessa Cell Analyzer User s Guide Fj Edit Statistics View Hender Populations Sats e Click OK 11 Print the analysis Chapter 6 Recording and analyzing data 93 Your global worksheet analysis objects should look like the following Beads 001 Singlets PE positive Singlets 50 100 150 700 2450 FSC A te 1 000 Beads 001 Singlets Ti aii _ PercP Cy5 5 positive iat at wi i 1 oo a L pak m Th Tube Beads_001 Population Events Parent Total W all Events 10 000 100 0 Singlets 9675 96 8 96 8 FITC positive 4 rrd 49 3 477 PE positive 1 499 15 5 15 0 Fer FP Cy5 5 positive 1 529 15 8 15 3 APC positive 1 629 1
20. remain on the SIP to prevent salt deposits from forming in the injection tube This tube also catches back drips from the flow cell Caution Do not leave more than 1 mL of water on the SIP When the instrument is turned off or left in standby mode a small amount of fluid will drip back into the sample tube If there is too much fluid in the tube it could overflow and affect the cytometer performance Chapter 4 Maintenance 43 Shutting down the cytometer About this topic This topic describes how to shut down the cytometer Before you begin Each time you shut down the cytometer perform the daily cleaning as described in Cleaning the fluidics page 41 Procedure To shut down the cytometer 1 Place a tube of DI water on the SIP 2 Turn off the flow cytometer 3 Select Start gt Shutdown to turn off the computer if needed 4 If your system is using the BD FACSFlow supply system shut off the BD FACSFlow supply system If the cytometer will not be used for a week or longer perform a system flush and leave the fluidics system filled with DI water to prevent saline crystals from clogging the fluidics Related topics e Cleaning the fluidics page 41 e Flushing the system page 44 44 BD LSRFortessa Cell Analyzer User s Guide Flushing the system About this topic Cautions A A Procedure This topic describes how to perform an overall fluidics cleaning to remove debris and contaminants from the she
21. run a performance check 1 Select Cytometer gt CST Cytometer Setup and Tracking File Cytometer Tools Setup Reports Performance Tracking System Summary Requires Attention Setup Control Research Use Only Load a tube with beads and click Run button to start setup Cytometer Configuration 6 Blue 4 Red 6 Violet 2 UV BD default Lot ID Characterize Check Performance Cytometer Configuration 6 Blue 4 Red 6 Violet 2 U BD t Cytometer Baseline January 18 2010 10 26 AM Cytometer Performance lt b Select Configuration No Cytometer Performance is available for current configuration and bead lot Setup Beads Lot ID 54102 Product CST Setup Beads Part 910723 Expiration Date 09 30 2011 Parameter Connected 2 Verify that the bead lot information under Setup Beads matches the Cytometer Setup and Tracking bead lot Setup Beads Lok ID 74102 we Product CST Setup Beads Fart W10723 Expiration Date 09 30 2011 66 BD LSRFortessa Cell Analyzer User s Guide 3 Verify that the cytometer configuration is correct for your experiment System Summary OK If the cytometer is not set to the correct configuration a Click Select Configuration in the Setup Control window b Select the correct configuration from the list c Click Set Configuration and then click OK 4 Verify that the current configuration has a
22. settings 169 3 In the Inspector click the Parameters tab and select the H checkbox to select height for each parameter Ea Cytomeler 5A100004 Pilot 4 LSRFortessa 1 ooj P gals kaika oaaao AE JERI T AE se Ja s m JHE ama F a Gis sila ils EE Es ra ca ca ka es EE apana apanas aeee seee E E al e oopebop gt oop ooboop lt i 4 On the global worksheet create the following plots and histograms FSC vs SSC dot plot FSC H and FSC A histogram FITC H and FITC A histogram APC H and APC A histogram 5 Create a P1 gate in the FSC vs SSC plot and show only the P1 population in all histograms 170 BD LSRFortessa Cell Analyzer User s Guide 6 Create three statistic views showing the following FSC H and FSC A means for P1 FITC H and FITC A means for P1 APC H and APC A means for P1 Your worksheet should look similar to the following figure Sphtimen O00 Tube Ot Tube Name Tube Ji Tube Hame Tubo_o01 Tebo Mame Tue Ni Record Gate Record Date Record Date PGCA FoC M FITC ETCH Posulabsn Migi Mein Pointiin Missin H in Popelaaces Et Free FREE FEFE FERF Hr Chapter 10 Manual settings 171 7 Expand the new specimen then set the current tube pointer to tube_001 8 Install the FITC positive control tube onto the loading port and click Load in the Acquisition Dashboard 9 Adjus
23. step 6 Preserve the setting that maximizes the fluorescence intensity 10 Reset the Window Extension to 10 ps 168 BD LSRFortessa Cell Analyzer User s Guide Adjusting area scaling About this topic About area scaling About this example Adjusting area scaling This topic describes how to manually adjust the area scaling on your cytometer if necessary for your application The area scaling is automatically set in CS amp T Depending on the size of your target particle you might need to adjust the area scaling manually Larger particles are more likely to require an area scaling adjustment The area of a pulse is calculated by BD FACSDiva using measured height and width measurements It is sometimes important to verify that the area calculation and the height measurement are equivalent by adjusting the factor applied to the area The required area scaling factor changes based on sheath pressure and particle size The following example describes how to adjust area scaling for an experiment which uses only the 488 nm laser and the 640 nm laser You must adjust area scaling for all lasers used in your experiment To adjust the other lasers add a parameter and the corresponding plots from that laser to the procedure To adjust area scaling 1 Open an existing experiment or create a new experiment in the Browser 2 Create a new specimen by clicking the New Specimen button on the Browser toolbar Chapter 10 Manual
24. the gray boxes Align the center of the negative population with the crosshair visible in the gray box Note Do not decrease the fluorescence PMT voltages Doing so can make it difficult to resolve dim populations from the negative population 4 Unload the unstained control tube from the cytometer 5 Load the multicolor sample onto the cytometer or load single color control tubes and verify each fluorochrome signal separately Chapter 5 Optimizing cytometer settings 75 6 Verify that the positive populations are on scale If a positive population is off scale lower the PMT voltage for that parameter until the positive population can be seen entirely on scale a Unload the multicolor sample 8 Place a tube containing DI water on the SIP and put the cytometer on standby m Optional Save the application settings by right clicking Cytometer settings in the Browser then selecting Application Settings gt Save 10 In the Save Application Settings dialog enter a descriptive name for the application settings Save Application Settings Enter a name For the Application Settings LSRFortessa 4 Color Application Settings 11 Click OK The application settings are saved to the catalog 76 BD LSRFortessa Cell Analyzer User s Guide Recording compensation controls About this topic This topic describes how to create and record compensation controls using the Compensation Setup feature of BD FACSDiv
25. 127 26 BD LSRFortessa Cell Analyzer User s Guide Preparing the sheath container About this topic This topic describes how to prepare the sheath container Note If your system is using the BD FACSFlow supply system please see the documentation provided with your system When to check the sheath Check the fluid levels in the sheath container every time you container use the cytometer This ensures that you do not run out of sheath fluid during an experiment Sheath container components Clamp knob Cap handle Air line green Tank handle Vent valve Sheath fluid line blue to cytometer Filter assembly Procedure Chapter 3 Cytometer setup 27 To prepare the sheath container 1 Verify that the flow cytometer is in standby mode Press the STNDBY button on the control panel if necessary Disconnect the air line green from the sheath container Depressurize the sheath container by pulling up on the vent valve Remove the sheath container lid Unscrew the clamp knob and push down to loosen if necessary Tilt the cap to the side to remove it from the tank Add 6 L of sheath fluid such as BD FACSFlow solution to the sheath container Caution Do not fill the sheath tank to its maximum capacity 8 L When an overfull tank is pressurized erratic cytometer performance can result Replace the sheath container lid Reconnect the air line green Make sure the gasket on the inside
26. 6 Five color blue laser configuration page 140 Three color red laser configuration page 143 Six color violet octagon page 147 Number of lasers 4 Colors 10 13 16 18 Lasers 4 Blue 2 Red 2 Violet 2 UV 5 Blue 3 Red 3 Violet 2 UV 5 Blue 3 Red 6 Violet 2 UV 6 Blue 4 Red 6 Violet 2 UV 136 BD LSRFortessa Cell Analyzer User s Guide See section Four color blue laser configuration page 139 Two color red trigon configuration page 142 Two color violet laser configuration page 145 Two color UV laser configuration page 148 Five color blue laser configuration page 140 Three color red laser configuration page 143 Three color violet laser configuration page 146 Two color UV laser configuration page 148 Five color blue laser configuration page 140 Three color red laser configuration page 143 Six color violet octagon page 147 Two color UV laser configuration page 148 Six color blue laser configuration page 141 Four color red laser configuration page 144 Six color violet octagon page 147 Two color UV laser configuration page 148 Chapter 9 Detector array configurations 137 Base configuration The BD LSRFortessa has one base configuration at installation Custom configurations can be added for different applications The following image shows a default base cytometer configuration Cytometer Configuration Cytometer BD LSR Fortessa Cytometer Name LSRFortes
27. 8 9 18 3 Specimen Name FourColorBeads Tube Mame Beads 001 FITC A PE A PerCP cy5 5 A Population Mean Mean Mean Wi Singlets 1 969 1 727 1 257 P FITC positive 3 880 40 MB FE positive g5 10 858 M PercP cy5 5 positive 106 BB APC positive g1 59 94 BD LSRFortessa Cell Analyzer User s Guide Related topics e Reusing an analysis page 94 Reusing an analysis About this topic Reusing an analysis Saving the analysis This topic describes how to use a global worksheets to apply the same analysis to a series of recorded tubes Once you define an analysis for a tube you can use it to analyze the remaining tubes in the experiment After viewing the data print the analysis or save it to a normal worksheet To reuse the analysis 1 Set the current tube pointer to the Beads_002 tube 2 View the Beads_002 data on your analysis worksheet Adjust the gates as needed Adjustments apply to subsequent tubes viewed on the worksheet To avoid altering a global worksheet save an analysis to a normal worksheet then make adjustments on the normal worksheet 3 Print the analysis When you perform analysis with a global worksheet the analysis does not save with the tube If you define your analysis on a global worksheet before recording data you can specify to automatically save the analysis after recording data You set this option in User Preferences To save a copy of the analysis with a tube 1 Expa
28. BD LSRFortessa Cell Analyzer User s Guide For Research Use Only AM bdbiosciences com 23 11093 00 Rev A 3 2010 Becton Dickinson and Company BD Biosciences BD Biosciences European Customer Support San Jose CA 95131 Tel 32 2 400 98 95 Tel 877 232 8995 Fax 32 2 401 70 94 Fax 800 325 9637 help biosciences europe bd com ResearchApplications bd com Copyrights 2010 Becton Dickinson and Company All rights reserved No part of this publication may be reproduced transmitted transcribed stored in retrieval systems or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without prior written permission from BD Biosciences The information in this guide is subject to change without notice BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments Although this guide has been prepared with every precaution to ensure accuracy BD Biosciences assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information BD Biosciences welcomes customer input on corrections and suggestions for improvement Trademarks Clorox is a registered trademark of The Clorox Company SPHERO is a trademark of Spherotech Inc Modfit LT is a trademark of Verity Software House Inc Microsoft and Windows are registere
29. Customers outside the US and Canada contact your local BD representative or distributor Need more information fast Information about this product is also available in your software s Help system You can keep the Help window open while you use the software or print the information directly from the window Internet access is not required to use the Help system The following topics are covered in this section e Instrument overview page 10 e Components page 11 e Fluidics page 13 e Sheath and waste containers page 17 e Optics page 18 e Workstation page 21 Introduction 10 BD LSRFortessa Cell Analyzer User s Guide Instrument overview The BD LSRFortessa is an air cooled multi laser benchtop flow cytometer with the ability to acquire parameters for a large number of colors It uses fixed alignment lasers that transmit light through a flow cell to configurable octagon and trigon detector arrays These detectors collect and translate the resulting fluorescence signals into electronic signals Cytometer electronics convert these signals into digital data Chapter 2 Introduction 11 Components About this topic This topic describes the instrument s components Instrument overview e o Heat ventilation slots Power switch right side ortess4 e BD LSRF Sample injection port SIP Front doors trigon detector arrays Side drawer octagon or trigon Control panel detector arra
30. D default Blue Laser Red Laser Violet Laser UY Laser 488nm 640nm 405nm 355nm Custom Configurations FSC al Blue Red Violet uv Window Extension us 10 00 62 BD LSRFortessa Cell Analyzer User s Guide Your cytometer might include only the base configuration when your cytometer is installed You can create additional configurations later as needed In this example the cytometer configuration must include the following parameters FITC PE PerCP Cy5 5 and APC 2 If you need to select a configuration other than the current configuration a In the Configurations tab select a configuration b Click Set Configuration c Click OK d Verify that the configuration you just set matches your BD LSRFortessa cytometer optics 3 Click OK to close the Cytometer Configuration window 4 Select File gt Exit to close CS amp T 5 Select Edit gt User Preferences Chapter 5 Optimizing cytometer settings 63 6 Click the General tab and select the Load data after recording checkbox Fj User Preferences General Gates Worksheet Plot FCS Templates Statistics Biexponentiall C Tube specific worksheet F Start acquisition on pointer change C Show file identifier GUID in statistics view C Remove tube specific cytometer settings on duplicate Save analysis after recording through global worksheet See the BD FACSDiva Software Reference Manual
31. Instrument is not warmed up Recommended solutions Prime the fluidics system See Priming the fluidics on page 33 Verify that sample remains in the tube and if necessary add sample to the tube or install a new sample tube Mix the sample to suspend the cells Empty the waste tank Adjust the PMT voltages Increase the number of events to display Remove the sample tube to allow backflushing If the event rate is still erratic clean the sample injection tube See Cleaning the fluidics on page 41 Replace the Bal seal See Changing the Bal seal on page 52 Wait 30 minutes for the instrument to warm up No events in acquisition display and RUN button is orange Possible causes Laser delay is set incorrectly Laser is not functioning Tube is cracked or misshapen Possible causes RUN is not activated Sample tube is not installed or is not properly seated Waste container is pressurized Sample tube is cracked Waste tubing line is not connected to the waste cap Chapter 8 Troubleshooting 119 Recommended solutions Adjust the laser delay settings See Manual settings on page 163 Verify the malfunction by changing the threshold to an alternative laser while running the appropriate sample If unsuccessful contact BD Biosciences Replace the sample tube Recommended solutions Press the RUN button Install the sample tube correctly on the SIP Replace the waste air fil
32. No signal in red laser channels High event rate Possible causes Incorrect laser delays due to a change in the sheath tank fluid level Possible causes Air bubbles in the sheath filter or flow cell Threshold level is too low PMT voltage for the threshold parameter is set too high Sample is too concentrated Sample flow rate is set to HI Chapter 8 Troubleshooting 121 Recommended solutions Check the fluid level in the sheath tank and refill if necessary e Adjust the laser delay settings See Manual settings on page 163 Recommended solutions Remove the air bubbles See Removing air bubbles on page 28 Increase the threshold level See the BD FACSDiva Software Reference Manual for instructions Set the PMT voltage lower for the threshold parameter See the BD FACSDiva Software Reference Manual for instructions Dilute the sample Set the sample flow rate to MED or LO 122 BD LSRFortessa Cell Analyzer User s Guide Low event rate Possible causes Threshold level is too high Air bubble or debris in the flow cell PMT voltage for the threshold parameter is set too low Sample is not adequately mixed Sample is too diluted Sample injection tube is clogged Recommended solutions Lower the threshold level See the BD FACSDiva Software Reference Manual for instructions Prime the fluidics system See Priming the fluidics on page 33 Set the PMT voltage hi
33. O EASES 44 Maintaining the waste management system 0 0 ce cee eee ee eee 46 Replace thie waste alr filter sasadan Bos pace Geo kOe wo Se EO eae 49 Changing the sheath Miter 5 1548 Seah AeA AWE RESTA CLES 50 Changinotie Balsseal irrena bak Giclee eeeas Saw ee new eee a 52 Changing the sample tube O ring 0 0 cece eee teens 54 Chapter 5 Optimizing cytometer settings 57 Cytometer settings workflow 2 0 0 eee etn ees 58 Verifying the configuration and user preferences 0 00 e nnna 61 Runnine a performance Checka acs 84w 5544 an a does deo RMR 64 Seine UP aex PEEN 24 62425054 1 8o oe bah ae otek sak ae 69 Creatine ap plication Setting S orereta re e VEE ae ee eee de pase 73 Recording compensation controls 0 ccc ee eee eee eee neee 76 Calculating compensation lt i 0 2 eds arsed 6 eb wae ee AS EE SE OOS 80 Chapter 6 Recording and analyzing data 81 Data recording and analysis workflow 4 44 34 oan Gri aoa Me Pe ew wees 82 Preparing the workspace saws aati ee 4 eee aes Se ae wea ee 83 Recording data tone GES ee eek EE Ee eh AO ENA EA ee SEGARA 85 Anal VAn Cala aad corey ee vue a E we eed Scores a Gerke OS RS 88 ROUSING Aira aly SIS ooien aer a Pecans a e E E eee 94 Chapter 7 Technical overview 97 ADOUtTMUGICS cious bee a raa a ea a aaa ahd aes 98 ADOM ODIS 2 3 ce nad beast eS E a E Saw Res ned a ae eee 100 ABOU ClECIEOMICS aeree e Baia hod eons Ae ed Ch eke ee heen de 111 Contents vii Cha
34. PE Cy7 780 60 Chapter 9 Detector array configurations 153 Green 532 nm 150 mW The fluorochromes listed in the following table can be used with this laser wavelength for the cell surface markers application See Common green laser configuration page 162 for a common octagon configuration map Fluorochromes BP filters PE 575 25 PE Texas Red 610 20 PE Cy5 660 20 670 30 PE Cy5 5 695 40 710 50 PE Cy7 780 60 154 BD LSRFortessa Cell Analyzer User s Guide Common blue laser The following maps show two common configurations for configurations the 488 nm blue laser PerCP Cy5 5 SSC PE Texas Red Chapter 9 Detector array configurations 155 PerCP Cy5 5 SSC Note that with the 488 nm blue laser FITC requires a 525 50 notch filter if the cytometer also uses a 532 nm laser 156 BD LSRFortessa Cell Analyzer User s Guide Common red laser The following maps show two common configurations for configurations the 640 nm red laser APC Alexa Fluor 700 gt Cy7 SS Cy7 Chapter 9 Detector array configurations 157 Common violet laser The following maps show two common configurations for configurations the 405 nm violet laser V450 or Pacific Blue gt gt AmcCyan 158 BD LSRFortessa Cell Analyzer User s Guide Qdot 700 or Qdot 705 Qdot 605 Qdot 656 AmCyan V450 or Pacific Blue Qdot 585 Qdot 800 Qdot 655 Chapter 9 Detector a
35. Procedure To optimize laser delay 1 While acquiring data from your sample create a histogram to show the fluorescence signal excited by the laser for which the delay is to be optimized 2 Inthe Acquisition Dashboard set the Events to Display to 500 events 3 Click the Laser tab in the Cytometer window 166 BD LSRFortessa Cell Analyzer User s Guide Window extension and laser delay values are displayed in microseconds Is me ey ee Se ee u a S al P ri Cyt OCS Orne Cen 4 Set the Window Extension value to 0 ps 5 Set an initial laser delay value only for the laser you are optimizing If you are optimizing the violet laser set its delay to 40 ps If you are optimizing the UV laser set its delay to 75 ps If you are optimizing the red laser set its delay to 110 ps Chapter 10 Manual settings 167 6 While observing the positive events on the histogram adjust the laser delay in 1 ys increments You might need to adjust the delay above or below the initial setting Choose the setting that moves the events farthest to the right highest fluorescence intensity 7 Draw an interval gate on the histogram for the positive events 8 Create a statistics view to display the mean fluorescence intensity MFI of the gated population 9 While observing the MFI for the gated population adjust the laser delay in 0 1 1s increments within a range of 2 0 ps of the setting obtained in
36. a software and an experiment with optimized settings Creating compensation To create compensation control tubes tubes 1 Select Experiment gt Compensation Setup gt Create Compensation Controls The Create Compensation Controls dialog opens For this bead example you do not need to provide non generic tube labels E Modify Compensation Controls e PerlP Cyo 5 Recording compensation settings Chapter 5 Optimizing cytometer settings 77 2 Click OK Compensation control tubes are added to the experiment Worksheets containing appropriate plots and gates are added for each compensation tube EA Compensation Cont ce S Cytometer Sett J Unstained Cont FITC Stained Ce PE Stained Con J PercP cy5 5 St APC Stained Co To record compensation settings 1 23 Press RUN and HI on the cytometer fluid control panel Install the unstained control tube onto the SIP Expand the Compensation Controls specimen in the Browser Set the current tube pointer to the unstained control tube it becomes green then click Acquire Data in the Acquisition Dashboard Verify that the population of interest is displayed appropriately on the FSC vs SSC plot and adjust voltages if necessary Unstained Control oe a Sa a i EE a 200 250 78 BD LSRFortessa Cell Analyzer User s Guide 10 11 12 Since the application settings have been optimized for your sample the c
37. arts 176 O ring ordering 179 replacing 54 worn 116 P particles 177 PerCP Stokes shift 101 performance check 64 65 photodiode 20 photomultiplier tubes PMTs 20 PMT See photomultiplier tubes population hierarchy 88 power switch 11 12 PRIME fluid control button 14 priming fluidics system 33 propidium iodide PI 41 pulse definition 111 measurements 112 processors 112 Q quality control QC particles 177 troubleshooting 125 R recording compensation settings 77 compensation tubes 76 data 82 85 red 640 nm laser 142 144 150 156 removing air bubbles filter 28 replacing Bal seal 52 battery 47 optical filters 36 sample tube O ring 54 sheath filter 50 waste air filter 49 reusing analyses 94 rinsing solution 42 RUN button orange 119 fluid control button 14 S safety symbols 3 sample optimization 58 optimization experiment 69 optimization single stained controls 60 82 SAMPLE FINE ADJ knob 13 sample injection port SIP components 15 hydrodynamic focusing 98 location 11 replacing Bal seal 52 replacing sample tube O ring 54 sample tube injection 15 not fitting 117 requirements 117 samples running 85 Index 185 saving analyses 94 scatter light 100 parameter distortion 123 setup cytometer 58 sheath flow 98 pressure 98 sheath container components shown 26 defined 17 depressurize 27 preparing 27 sheath filter components shown 50 ordering 179 removing air bubbles 28 replacing 50 s
38. ath tubing waste tubing and flow cell Perform the system flush at least every 2 weeks Note If you are using the BD FACSFlow supply system see the BD FACSFlow Supply System User s Guide for instructions on flushing the system Caution Biohazard The cytometer hardware might be contaminated with biohazardous material Use 10 bleach to decontaminate the instrument To perform a system flush 1 Remove the sheath filter a Press the quick disconnects on both sides of the filter assembly b Remove the filter assembly c Connect the two fluid lines Caution Do not run detergent bleach or ethanol through the sheath filter They can break down the filter paper within the filter body causing particles to escape into the sheath fluid possibly clogging the flow cell 2 Empty the sheath container and rinse it with DI water 3 Fill the sheath container with at least 1 L of undiluted BD FACSClean solution 4 Empty the waste container if needed 5 Open the roller clamp by the fluidics interconnect and drain the fluid into a beaker for 5 seconds Next step 10 11 12 13 Chapter 4 Maintenance 45 Remove the DI water tube from the SIP Prime the instrument twice a Press the PRIME button on the fluidics control panel b When the STNDBY button lights amber press the PRIME button again Install a tube with 3 mL of undiluted BD FACSClean solution on the SIP and put the tube support arm un
39. aths For example Select File gt Print means to select the Print option from the File menu When used with key names a plus sign means to press two keys simultaneously For example Ctrl P means to hold down the Control key while pressing the p key Chapter 1 About this guide 5 About the BD LSRFortessa documentation About this topic This topic describes the documentation available with the BD LSRFortessa flow cytometer Publication formats This guide is provided in PDF format to provide an eco friendly option All content is also included in the BD FACSDiva software Help Online help The online help installed with BD FACSDiva software includes all content from this guide and the documents listed below Access BD LSRFortessa online help from the Help menu in BD FACSDiva software The online help is compiled from the following documents e BD FACSDiva Software Reference Manual Includes instructions or descriptions for installation and setup workspace components acquisition controls analysis tools and data management Access this manual from the BD FACSDiva Software Help menu Help gt Literature gt Reference Manual or by double clicking the shortcut on the desktop e BD Cytometer Setup and Tracking Application Guide Describes how to use the Cytometer Setup and Tracking features in BD FACSDiva software e BD LSRFortessa Cell Analyzer Site Preparation Guide Contains specifications for Cytomet
40. ble and the data is similar to the previous sample 12 Click Record Data 13 When event recording has completed remove the second tube from the cytometer Related topics 14 15 16 17 Chapter 6 Recording and analyzing data 87 If you are recording more than two tubes repeat steps 8 through 13 for the remaining tubes Print the experiment level cytometer settings by right clicking the Cytometer Settings icon in the Browser and selecting Print Install a tube of DI water onto the SIP Place the cytometer in standby mode Analyzing data page 88 88 BD LSRFortessa Cell Analyzer User s Guide Analyzing data About this topic Analyzing data This topic describes how to analyze recorded tubes by creating plots gates a population hierarchy and statistics views on a new global worksheet To analyze data 1 Use the Browser toolbar to create a new global worksheet Rename it MyDataAnalysis Create the following plots on the MyDataAnalysis worksheet FSC vs SSC FITC vs PE FITC vs PerCP Cy5 5 FITC vs APC Create a population hierarchy and a statistics view and set them below the plots on the worksheet Right click any plot and select Show Population Hierarchy Right click any plot and select Create Statistics View Set the current tube pointer to Beads_001 Draw a gate around the singlets on the FSC vs SSC plot Use the population hierarchy to rename the pop
41. d of a mirror or filter A dash indicates that no slot exists for a mirror in that PMT position Chapter 9 Detector array configurations 139 Four color blue laser The following map shows the four color configuration for configuration the 488 nm blue laser PMT LP mirror BP filter Fluorochromes A 750 780 60 PE Cy 7 B 685 695 40 PerCP Cy5 5 PE Cy 5 PerCP PI 550 575126 PE 505 530 30 FITC Alexa Fluor 488 Blank 488 10 SSC 140 BD LSRFortessa Cell Analyzer User s Guide Five color blue laser The following map shows the five color configuration for the configuration 488 nm blue laser PMT LP mirror BP filter Fluorochromes A 750 780 60 PE Cy7 B 685 695 40 PerCP Cy5 5 C 600 610 20 PE Texas Red D 550 575126 PE E 505 530 30 FITC Alexa Fluor 488 F Blank 488 10 SSC Six color blue laser configuration Chapter 9 Detector array configurations 141 The following map shows the six color configuration for the 488 nm blue laser PMT A B C D E F G LP mirror 750 685 655 600 550 505 Blank BP filter 780 60 695 40 670 30 610 20 575 26 530 30 488 10 Fluorochromes PE Cy7 PerCP Cy5 5 PE CyS PE Texas Red PE FITC Alexa Fluor 488 SSC 142 BD LSRFortessa Cell Analyzer User s Guide Two color red trigon The following map shows the two color configuration for configuration the 640 nm red laser PMT LP mirror BP filter Fluoroch
42. d trademarks of Microsoft Corporation Texas Red and Alexa Fluor are registered trademarks and Pacific Blue is a trademark of Molecular Probes Inc Teflon is a registered trademark of E I du Pont de Nemours and Company Cy is a trademark of Amersham Biosciences Corp Cy dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use Any other use requires a commercial sublicense from Amersham Biosciences Corp 800 Centennial Avenue Piscataway NJ 08855 1327 USA BD BD Logo and all other trademarks are property of Becton Dickinson and Company 2010 BD Patents The BD LSRFortessa Cell Analyzer is covered by one or more of the following US Patents and foreign equivalents 7 129 505 6 944 338 6 809 804 6 683 314 6 014 904 APC Cy7 US 5 714 386 BD FACS lysing solution US 4 654 312 4 902 613 5 098 849 Regulatory information For Research Use Only Not for use in diagnostic or therapeutic procedures Class I 1 Laser Product 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
43. derneath the tube See Maintenance overview page 40 for other recommended cleaning solutions Press RUN and HI on the cytometer fluid control panel Run for 30 minutes Press the STNDBY fluid control button and depressurize the sheath container by lifting the vent valve Repeat steps 2 through 10 with BD FACSRinse solution Repeat steps 2 through 10 with DI water Replace the sheath filter and refill the sheath container with sheath fluid Test the waste management system battery as described in Maintaining the waste management system page 46 46 BD LSRFortessa Cell Analyzer User s Guide Maintaining the waste management system About this topic This topic describes how to maintain the waste management system The waste management system has an alarm powered by a 9 volt battery that you must test and change regularly to ensure continued operation When to perform the Test the battery every two weeks after you flush the system battery test Change the battery as needed Testing the battery and To test the battery and alarm alarm l 1 Locate the Battery Test switch on the waste container bracket Battery test switch BATTERY TEST 2 Toggle the switch If the battery and the alarm are working properly you should hear an alarm buzzing If you do not hear any sound change the battery as described in the following section 3 Release the switch Chapter 4 Maintenance 47 Changing the
44. e Related topics This topic describes how to verify that the optical filters are in the appropriate position for your particular requirements Before you run samples you must set up the optical filters Caution Laser Hazard Follow the precautions outlined in the BD LSRFortessa Safety and Limitations Guide while changing optical filters or mirrors To change a filter or mirror 1 Access the appropriate detector array Octagon or trigon arrays are located in the left side cytometer drawer Up to twotrigon arrays are located in the front doors 2 Remove the appropriate filter holder or mirror holder 3 Replace the removed holder with the new filter or mirror holder The metal ring on the filter or mirror holder must face toward the center of the trigon or octagon Verify that the filters are arranged so that the longest wavelength is in the A position and the shortest wavelength is in the last position used 4 Close the cytometer side drawer or front door s e Detector array configurations page 129 Chapter 3 Cytometer setup 37 Custom configurations and baselines About this topic Overview Related topics This topic describes where to find information on how to create a custom configuration and define a baseline for a performance check BD Cytometer Setup and Tracking CS amp T software is used to define the baseline performance of your cytometer A baseline provides a starting point
45. e 54 40 BD LSRFortessa Cell Analyzer User s Guide Maintenance overview About this topic General use guidelines A A This topic provides an overview of the BD LSRFortessa routine maintenance and cleaning procedures Caution Biohazard Coming into contact with all biological specimens and materials is considered biohazardous Follow these guidelines whenever operating or maintaining the cytometer e Handle all biological specimens and materials as if they are capable of transmitting infection e Dispose of waste using proper precautions and in accordance with local regulations e Never pipette by mouth e Wear suitable protective clothing eyewear and gloves For fluidics maintenance we recommend the following cleaning solutions e BD FACSClean solution e 10 bleach solution e 0 5 solution of sodium hypochlorite Use DI water to dilute bleach and sodium hypochlorite to appropriate concentrations Caution Higher concentrations of sodium hypochlorite and use of other cleaning solutions might damage the cytometer When to perform maintenance procedures Chapter 4 Maintenance 41 Perform maintenance procedures in the following frequencies Frequency Daily Scheduled every two weeks Periodic frequency depends on how often you run the cytometer Cleaning the fluidics About this topic Overview Maintenance procedure Cleaning the fluidics page 41 Shutting down the cyt
46. e Browser changes Creating application settings page 73 Recording data page 85 Recording data Chapter 6 Recording and analyzing data 85 About this topic This topic provides an example of how to preview and record data for multiple samples Before you begin Prepare the sample tubes Recording data To record data 1 Press RUN and HI on the cytometer fluid control panel 2 Install the first sample tube onto the SIP 3 Set the current tube pointer to Beads_001 4 Click Acquire Data in the Acquisition Dashboard to begin acquisition 5 While data is being acquired Draw a gate around the singlets on the FSC vs SSC plot Rename the P1 gate to Singlets Use the Inspector to set the other plots to show only the singlet population by selecting the Singlets checkbox E All Everts M B inget 1 Click Record Data 86 BD LSRFortessa Cell Analyzer User s Guide 7 When event recording has completed remove the first tube from the cytometer The MyData worksheet plots should look like the following FourColorBeads Beads 001 FourColorBeads Beads 001 50 i00 1450 200 250 258769 125946 FSC A fe 1 000 FourColorBeads Beads 001 PercP Cy5 5 8 Install the second sample tube onto the SIP 9 Set the current tube pointer to Beads_002 10 Click Acquire Data to begin acquisition 11 Before recording preview the data on the MyData worksheet to verify that all expected populations are visi
47. e tube is installed The SIP includes the sample injection tube and the tube support arm Samples are introduced through a stainless steel injection tube equipped with an outer droplet containment sleeve The sleeve works in conjunction with a vacuum pump to eliminate droplet formation of sheath fluid as it backflushes from the sample injection tube Outer sleeve Sample injection tube Tube support arm e Sample injection tube Stainless steel tube that carries sample from the sample tube to the flow cell This tube is covered with an outer sleeve that serves as part of the droplet containment system e Tube support arm Arm that supports the sample tube and activates the droplet containment system vacuum The vacuum is on when the arm is positioned to the side and off when the arm is centered Note If a sample tube is left on the SIP with the tube support arm to the side vacuum on the sample will be aspirated into the waste container 16 BD LSRFortessa Cell Analyzer User s Guide Cautions when using the HTS option A A A A Droplet containment module Caution Biohazard When using the BD LSRFortessa cytometer with the BD High Throughput Sampler HTS ensure that the HTS is completely pushed into the operating position before removing the DCM droplet containment module sleeve or disconnecting the sample coupler from the SIP This is to avoid accidental leakage of potentially biohazardous liquids direct
48. elay page 165 Troubleshooting This chapter covers the following topics e Cytometer troubleshooting page 116 e Electronics troubleshooting page 127 116 BD LSRFortessa Cell Analyzer User s Guide Cytometer troubleshooting About this topic Droplets are visible on the SIP This topic describes possible problems and recommended solutions for BD LSRFortessa cytometer issues Possible causes Worn O ring in retainer Outer sleeve is not seated in the retainer Outer sleeve is not on the sample injection tube Waste line is pinched preventing proper aspiration Waste tank is full Droplet containment vacuum not functioning The HTS acquisition control switch is in plate mode Recommended solutions Replace the O ring See Changing the sample tube O ring page 54 1 Loosen the retainer 2 Push the outer sleeve up into the retainer until seated 3 Tighten the retainer Replace the outer sleeve 1 Loosen the retainer 2 Slide the outer sleeve over the sample injection tube until it is seated 3 Tighten the retainer Check the waste line Empty the waste tank Call your BD service representative Change the acquisition control switch to tube mode See the BD High Throughput Sampler User s Guide for more information Sample tube not fitting on SIP Rapid sample aspiration No events in acquisition display and RUN button is green Possible causes Sample tube ot
49. ems and recommended solutions for BD LSRFortessa electronic issues Possible causes Cytometer power is off Communication failure between workstation and cytometer Recommended solutions Turn on the cytometer main power e In BD FACSDiva software select Cytometer gt Connect e Ifconnecting does not work restart the cytometer Turn the cytometer off wait 1 minute and turn on the cytometer main power e If connecting still does not work contact BD Biosciences Need more information fast Information about this product is also available in your software s Help system You can keep the Help window open while you use the software or print the information directly from the window Internet access is not required to use the Help system Detector array configurations This chapter covers the following topics Fluorescence spectra page 130 About configuration maps page 132 About the base configuration page 133 Base configuration octagon and trigon maps page 138 Special Order configurations page 149 130 BD LSRFortessa Cell Analyzer User s Guide Fluorescence spectra About this topic Designing multicolor panels This topic shows sample emission spectra from common fluorochromes as well as the more common laser excitation lines This information is useful for designing multicolor panels An interactive fluorescence viewer is also available at bdbiosciences com colors fluorescence_spec
50. er weight and size Temperature and other environmental requirements Electrical requirements 6 BD LSRFortessa Cell Analyzer User s Guide e BD High Throughput Sampler User s Guide Describes how to set up and operate the BD High Throughput Sampler HTS option It also contains a description of BD FACSDiva software features specific to the HTS e BD FACSFlow Supply System User s Guide Describes the optional automated sheath and waste fluid control system designed for use with the BD LSR II and BD LSRFortessa Chapter 1 About this guide 7 Technical assistance About this topic This topic describes how to get technical assistance Getting assistance from For technical questions or assistance in solving a problem this guide e Read the sections of the documentation specific to the Operation you are performing See About the BD LSRFortessa documentation page 5 for more information e See Troubleshooting page 115 Contacting technical If additional assistance is required contact your local support BD Biosciences customer support representative or supplier When contacting BD Biosciences have the following information available e Product name part number and serial number e Version of BD FACSDiva software you are using e Any error messages e Details of recent system performance For cytometer support from within the US call 877 232 8995 For support from within Canada call 888 259 0187
51. escribed in Calculating compensation page 80 80 BD LSRFortessa Cell Analyzer User s Guide Calculating compensation About this topic Before you begin Procedure Related topics This topic describes how to calculate compensation Before you can calculate compensation you need to record the data for each single stained control To calculate compensation L Select Experiment gt Compensation Setup gt Calculate Compensation Note If the calculation is successful a dialog is displayed where you can enter a name for the compensation setup Note The default name is year month day time Enter a setup name and click Link amp Save The compensation is linked to the cytometer settings and saved to the catalog To help track compensation setups include the experiment name date or both in the setup name The compensation setup is linked to the MyExperiment cytometer settings and subsequent acquisitions in MyExperiment are performed with the new compensation settings We recommend that you always visually and statistically inspect automatically calculated spectral overlap values The means of the positive controls should be aligned with the means of the negative controls Recording compensation controls page 76 Recording and analyzing data This section includes the following topics Data recording and analysis workflow page 82 Preparing the workspace page 83 Recording data page 85
52. ew This section contains a technical overview of the following topics e About fluidics page 98 e About optics page 100 e About electronics page 111 98 BD LSRFortessa Cell Analyzer User s Guide About fluidics About this topic Pressure driven fluidics system Hydrodynamic focusing Low sample pressure 12 L min Sheath fluid Sample This topic describes the fluidics system in the BD LSRFortessa flow cytometer The fluidics system in the BD LSRFortessa flow cytometer is pressure driven A built in air pump provides a sheath pressure of 5 5 psi After passing through the sheath filter sheath fluid is introduced into the lower chamber of the quartz flow cell The sample to be analyzed arrives in a separate pressurized stream When a sample tube is placed on the SIP the sample is forced up and injected into the lower chamber of the flow cell by a slight overpressure relative to the sheath fluid The conical shape of the lower chamber creates a laminar sheath flow that carries the sample core upward through the center of the flow cell where the particles to be measured are intercepted by the laser beam This process is known as hydrodynamic focusing High sample pressure 60 uL min Laser beam Laser beam Sheath fluid Sheath fluid Sheath fluid Sample Chapter 7 Technical overview 99 The objective in flow cytometric analysis is to have at most one cell or particle
53. folder and rename it MyFolder Click MyFolder then click the New Experiment button on the Browser toolbar fe Browser MyExperiment jm lt Fie I E f Experiment T Name 44 Administrator Em MyFolder a Click the new experiment in the Browser and rename it MyExperiment 70 BD LSRFortessa Cell Analyzer User s Guide 5 Select MyExperiment in the Browser The Inspector displays details for the experiment Specifying parameters To specify the parameters for the new experiment 1 Select Cytometer Settings for the experiment in the Browser SLi MyExperiment 1 4 07 8 54 15 Cytometer Settings aE Global Worksheets Cytometer settings appear in the Inspector Fd Inspector Cytometer Settings Cytometer Settings Parameters Threshold Ratio Compensation oo ooz k T FITE FE PE Texos Red PE Cy5 PerCP Cy5 5 e PEAY APC Alexa Fluor 680 gaaaaat alak a a H BjejEE jE joo m m olola x Pudi Chapter 5 Optimizing cytometer settings 71 2 Make sure the parameters you need appear on the Parameters tab in the Inspector If more than one parameter is available for a particular PMT you might have to select the one you need from a menu For example you can set Detector F for the blue laser as FITC or Alexa Fluor 488 a Click the Parameter name to display the available fluorochromes in the Parameters list Inspec
54. for more information about cytometer configurations and user preferences Related topics e Setting up an experiment page 69 64 BD LSRFortessa Cell Analyzer User s Guide Running a performance check About this topic Overview Considerations Before you begin This topic describes how to run a performance check as part of quality control The CS amp T application is designed to monitor performance on a daily basis and to optimize laser delay Running a performance check on a regular basis provides a standard for monitoring changes in performance due to degradation of laser power aging of PMTs and other potential cytometer service issues Performance results are also affected by fluidics performance We strongly recommend following the fluidics maintenance procedures as described in Cleaning the fluidics page 41 Some BP filters might not be normalized to CS amp T settings In this case CS amp T will generate Qr and Br numbers that are not comparable from instrument to instrument They are however still trackable on one cytometer Part of the process for optimizing cytometer settings includes verifying PMT voltages set by CS amp T for all parameters Carefully examine any channel with a non CS amp T normalized filter Define the performance baseline for any configuration before running a performance check See Custom configurations and baselines page 37 Chapter 5 Optimizing cytometer settings 65 Procedure To
55. for the tracking of cytometer performance When running a performance check you compare the results to the baseline Some BP filters might not be normalized to CS amp T settings In this case CS amp T will generate Qr and Br numbers that are not comparable from instrument to instrument They are however still trackable on one cytometer In addition you must carefully check the PMT voltages that CS amp T sets for these filters See Optimizing cytometer settings page 57 Please see the latest published filter guides available on our website bdbiosciences com for more information See the BD Cytometer Setup and Tracking Application Guide for information on creating custom configurations and defining a baseline e Running a performance check page 64 Need more information fast Information about this product is also available in your software s Help system You can keep the Help window open while you use the software or print the information directly from the window Internet access is not required to use the Help system Maintenance This section covers the following topics e Maintenance overview page 40 e Cleaning the fluidics page 41 e Shutting down the cytometer page 43 e Flushing the system page 44 e Maintaining the waste management system page 46 e Replacing the waste air filter page 49 e Changing the sheath filter page 50 e Changing the Bal seal page 52 e Changing the sample tube O ring pag
56. full revolutions in either direction from its midpoint providing sample flow rates from 0 5 2X the midpoint value For example if the LO button is pressed the knob will give flow rates from approximately 6 24 pL min 14 BD LSRFortessa Cell Analyzer User s Guide Fluid control Three fluid control buttons RUN STNDBY and PRIME set the cytometer mode RUN Pressurizes the sample tube to transport the sample through the sample injection tube and into the flow cell The RUN button is green when the sample tube is on and the support arm is centered When the tube support arm is moved left or right to remove a sample tube the cytometer switches to an automatic standby status to conserve sheath fluid and the RUN button changes to orange STNDBY standby Stops fluid flow to conserve sheath fluid When you leave the cytometer for more than a few minutes place a tube containing 1 mL of deionized DI water on the sample injection port SIP and press STNDBY PRIME Prepares the fluidics system by draining and filling the flow cell with sheath fluid The fluid flow initially stops and pressure is reversed to force fluid out of the flow cell and into the waste container After a preset time the flow cell fills with sheath fluid at a controlled rate to prevent bubble formation or entrapment At completion the cytometer switches to standby mode Sample injection port Chapter 2 Introduction 15 The SIP is where the sampl
57. gher for the threshold parameter See the BD FACSDiva Software Reference Manual for instructions Mix the sample to suspend the cells Concentrate the sample If the flow rate setting is not critical to the application set the flow rate switch to MED or HI Remove the sample tube to allow backflushing If the event rate is still erratic clean the sample injection tube See Cleaning the fluidics on page 41 Erratic event rate Distorted scatter parameters Possible causes Sample tube is cracked Air bubble or debris in the flow cell Bal seal is worn Sample injection tube is clogged Contaminated sample Sheath filter is dirty Possible causes Cytometer settings are improperly adjusted Air bubble in sheath filter or flow cell Chapter 8 Troubleshooting 123 Recommended solutions Replace the sample tube Prime the fluidics system See Priming the fluidics on page 33 Replace the Bal seal See Changing the Bal seal on page 52 Remove the sample tube to allow backflushing If the event rate is still erratic clean the sample injection tube See Cleaning the fluidics on page 41 Prepare the specimen again Ensure that the tube is clean Replace the filter See Changing the sheath filter on page 50 Recommended solutions Optimize the scatter parameters See the BD FACSDiva Software Reference Manual for instructions Purge the air from the filter See Removing air bubble
58. h should be in the last position used There should not be any empty slots for any laser being used Always use a blank optic holder If a slot is filled with a filter or mirror an identifying number appears in that position on the configuration map If a slot is filled with a blank optic holder that position on the configuration map is unlabeled Chapter 9 Detector array configurations 133 About the base configuration About this topic Nine available configurations Number of lasers Colors 1 4 2 6 This section describes the base configuration options available with the BD LSRFortessa The base configuration for a BD LSRFortessa cytometer supports detectors filters and mirrors for up to four lasers to provide up to 18 color detection Lasers See section 4 Blue Four color blue laser configuration page 139 4 Blue Four color blue laser configuration page 139 2 Red Two color red trigon configuration page 142 5 Blue Five color blue laser configuration page 140 2UV Two color UV laser configuration page 148 5 Blue Five color blue laser configuration page 140 3 Red Three color red laser configuration page 143 134 BD LSRFortessa Cell Analyzer User s Guide Number of lasers 3 Colors 8 10 11 14 Lasers 4 Blue 2 Red 2 UV 4 Blue 2 Red 2 Violet 5 Blue 3 Red 2 UV 5 Blue 3 Red 3 Violet 5 Blue 3 Red 6 Violet See section Four color blue laser configuratio
59. han the PMTs A photodiode is used to detect the stronger forward scatter FSC signal However an optional PMT Chapter 2 Introduction 21 for detecting FSC is available through the BD Special Order Research Program Related topics e Optical filter theory page 103 e About the base configuration page 133 e Special Order configurations page 149 Workstation About this topic This topic describes the components of the BD LSRFortessa workstation Workstation components Acquisition analysis and most instrument functions are controlled by the BD LSRFortessa workstation It includes a PC one or two monitors and a printer Your workstation is equipped with the following e Microsoft Windows operating system e BD FACSDiva software version 6 2 or later for data acquisition and analysis e Software documentation including an online help system Related topics e About the BD LSRFortessa documentation page 5 Need more information fast Information about this product is also available in your software s Help system You can keep the Help window open while you use the software or print the information directly from the window Internet access is not required to use the Help system Cytometer setup This section includes the following topics Starting the cytometer and computer page 24 Preparing the sheath container page 26 Removing air bubbles page 28 Preparing the waste container page 30 Priming
60. heath fluid 98 backflush 16 BD FACSFlow sheath fluid 178 shortpass SP filters 103 105 side scatter SSC 100 signals amplifying 20 SIP See sample injection port software cytometer control 2 version 21 spare parts ordering 176 spillover 108 SSC See side scatter starting cytometer 24 statistics views 88 STNDBY fluid control button 14 Stokes shift 101 support technical 7 T technical assistance 7 testing battery 46 186 BD LSRFortessa Cell Analyzer User s Guide text conventions 4 Y thiazole orange TO 41 threshold defined 113 TO See thiazole orange trigon configuration maps 132 detector 10 location 36 troubleshooting cytometer 116 electronics 127 tubes Falcon 117 179 requirements 117 yellow green 561 nm laser 152 161 U user preferences 62 UV 355 nm laser 148 152 159 V violet 405 nm laser 145 147 151 157 W waste air filter component shown 31 replacing 49 waste air vent filter replacing 119 waste container 17 alarm 17 battery test 46 capacity 17 components shown 31 defined 17 emptying 30 pressurized 32 119 replacing battery 47 window extension 166 workstation about 21
61. heck the sheath line for air bubbles Open the roller clamp at the fluidics interconnect if necessary to bleed off any air in the line Collect any excess fluid in a waste container Close the roller clamp 30 BD LSRFortessa Cell Analyzer User s Guide Preparing the waste container About this topic A A When to check the waste container This topic describes how to prepare the waste container Note If your system is using the BD FACSFlow supply system please see the documentation provided with your system Caution Biohazard All biological specimens and materials coming into contact with them are considered biohazardous Handle as if capable of transmitting infection Dispose of waste using proper precautions and in accordance with local regulations Never pipette by mouth Wear suitable protective clothing eyewear and gloves Check the fluid levels in the waste container every time you use the cytometer This ensures that the waste container does not become too full Waste container components Cautions A A Chapter 3 Cytometer setup 31 Waste tubing orange from cytometer Waste air vent filter Level sensor line Bracket Caution Biohazard To avoid leakage of biohazardous waste put the cytometer in standby mode before disconnecting the waste container Caution Biohazard The waste container contents might be biohazardous Treat contents with bleach 10 of total volume 32
62. her than BD Falcon tubes used Worn Bal seal Sample tube is cracked Possible causes Support arm is to the side Droplet containment module is failing Possible causes Threshold is not set to the correct parameter usually FSC Threshold level is too high PMT voltage for threshold parameter is set too low Gating issue Air in the sheath filter Chapter 8 Troubleshooting 117 Recommended solutions Use BD Falcon 12 x 75 mm sample tubes See Equipment on page 179 Replace the Bal seal See Changing the Bal seal on page 52 Transfer contents to a new tube Recommended solutions Place the support arm under the sample tube Try the solutions in Droplets are visible on the SIP page 116 If the issue is not resolved call your BD service representative Recommended solutions Set the threshold to the correct parameter for your application Lower the threshold level Set the PMT voltage higher for the threshold parameter See the BD FACSDiva Software Reference Manual for information on setting gates Purge the filter See Removing air bubbles on page 28 118 BD LSRFortessa Cell Analyzer User s Guide Possible causes Air bubble or debris in flow cell No sample in the tube Sample is not mixed properly Waste tank is full PMT voltages set too low or too high for display parameter Too few events are displayed Sample injection tube is clogged Bal seal is worn
63. hod called compensation In the following example FITC emission appears primarily in the FITC detector but some of its fluorescence spills over into the PE detector The spillover must be corrected or compensated for Alternatively the spillover can be minimized by discrete excitation of fluorchromes In the following example excitation with a 561 nm laser special order will help minimize spillover FITC PE BP53080 BP57 5 26 Normalized intensity 500 nm 550 nm 600 nm 650 nm 700 nm Wavelength nm This spillover can be seen in a dot plot of FITC vs PE The FITC spillover in the PE detector must be corrected as demonstrated in the two figures that follow Chapter 7 Technical overview 109 eo FITC positive population Unstained particles FITC Using the Compensation tab of the Cytometer window in BD FACSDiva software you can adjust the PE FITC spectral overlap value Compensation is optimal when the positive and negative FITC populations have the same means in the PE parameter statistics The following image shows the FITC spillover optimally compensated out of the PE parameter PE A FITS positive Population Mean PE p Unlabeled 20 population Unstained E FITC positive 20 particles C ee Matching means FITC Once fluorescence compensation has been set for any sample the compensation setting remains valid for a subsequent dim or bright sample provided the signal is not saturated
64. hroic filters that are used to direct different color light signals to different detectors are called dichroic mirrors Although some of the properties of LP and SP filters are similar to dichroic mirrors for example allowing a specific wavelength range to pass filters and mirrors cannot be used interchangeably especially if used as dichroic mirrors A dichroic mirror must have a surface coating that reflects certain wavelengths but many LP or SP filters are absorbance filters that do not have any specific reflective characteristics Also optical filters and dichroic mirrors are rated at a specific angle of incidence When used in front of the fluorescence detectors they are perpendicular to the incident light and when used as a dichroic mirror they are placed at an angle relative to the light source Their optical properties are therefore designed for that angle of incidence 108 BD LSRFortessa Cell Analyzer User s Guide Compensation theory Fluorochromes emit light over a range of wavelengths Optical filters are used to limit the range of frequencies measured by a given detector However when two or more fluorochromes are used the overlap in wavelength ranges often makes it impossible for optical filters to isolate light from a given fluorochrome As a result light emitted from one fluorochrome appears in a detector intended for another This is referred to as spillover Spillover can be corrected mathematically by using a met
65. ing cytometer settings 60 BD LSRFortessa Cell Analyzer User s Guide About the examples The examples in this chapter use a 4 color bead sample with the following fluorochromes e FITC e PE e PerCP Cy 5S 5 e APC If you follow this workflow with a different bead sample or another sample type your software views data plots and statistics might differ from the example Additionally you might need to modify some of the instructions in the procedure The information shown in italics is for example only You can substitute your own names for folders and experiments Chapter 5 Optimizing cytometer settings 61 Verifying the configuration and user preferences About this topic This topic describes how to verify the cytometer configuration and user preferences before you create an experiment Caution To obtain accurate data results the current A cytometer configuration must reflect your BD LSRFortessa cytometer optics Procedure To verify the configuration and preferences before you create an experiment 1 Select Cytometer gt View Configurations and verify the current configuration Cytometer Configuration Cytometer BD LSR Fortessa Current Configuration Copy of 6 Blue 4 Red 6 iolet 2 UY Cytometer Name LSRFortessa SerialNumber 1 Configurations Parameters Filters and Mirrors 6 Blue 4 Red 6 Violet 2 UY BD default BS Base Configurations BE 6 Blue 4 Red 6 Violet 2 U B
66. ions 147 The following map shows the six color configuration for the 405 nm violet laser PMT A B D E F LP mirror 630 595 575 545 475 Blank BP filter 655 8 605 12 585 15 560 20 525 50 450 50 Fluorochromes Qdot 655 Qdot 605 Qdot 585 Qdot 565 AmCyan V500 Qdot 525 Pacific Blue V450 Marina Blue Alexa Fluor 405 148 BD LSRFortessa Cell Analyzer User s Guide Two color UV laser The following map shows the two color configuration for configuration the 355 nm UV laser PMT LP mirror BP filter Fluorochromes A SOS 530 30 Indo 1 Blue B Blank 450 50 Indo 1 Violet DAPI Alexa Fluor 350 Related topics e About the base configuration page 133 e Special Order configurations page 149 Chapter 9 Detector array configurations 149 Special Order configurations About this topic Available options BP filters used with CS amp T Blue 488 nm 20 100 mW This topic describes some of the more common configurations available through the Special Order Research Program The BD LSRFortessa cytometer can be configured with up to 5 lasers choosing from 11 different special order wavelength options and a variety of detectors This section shows some of the common special order laser detector combinations Contact your local BD Biosciences sales representative for information about other configurations Not all combinations of Special Order BP filters and laser
67. ly onto the instrument With the HTS in the proper location the containment dish with padding is directly below the SIP Caution If you are using the HTS option always slide the HTS mount slowly to prevent sample cross contamination when the wells are full Never move the HTS when it is in operation Caution Do not lean on or put any weight on the HTS as it could damage the instrument The DCM prevents sheath fluid from dripping from the SIP and provides biohazard protection When no sample tube is installed on the SIP sheath fluid backflushes through the sample injection tube This backflush helps prevent carryover of cells between samples The DCM vacuum is activated when the sample tube is removed and the tube support arm is moved to the side Sheath fluid is aspirated as it backflushes the sample injection tube Chapter 2 Introduction 17 Sheath and waste containers About this topic Sheath container Waste container Related topics This topic describes the sheath and waste containers The sheath and waste containers are outside the cytometer and are positioned on the floor Note If your system is using the BD FACSFlow supply system please see the documentation provided with your system The sheath container has a capacity of 8 L Sheath fluid is filtered through an in line interchangeable filter that prevents small particles from entering the sheath fluid lines Caution Do not fill the sheath tank to
68. mmend changing the sheath filter assembly every sheath filter 6 months Increased debris appearing in an FSC vs SSC plot can indicate that the sheath filter needs to be replaced See Supplies and consumables page 175 for ordering information Sheath filter components Sheath line Vent fitting Quick disconnect Vent line Filter base Removing the old filter Attaching the new filter Chapter 4 Maintenance 51 To remove the old filter 1 PA Place the cytometer in standby mode Remove the sheath filter assembly by pressing the quick disconnect on both sides of the filter assembly Over a sink or beaker Remove the vent line from the filter and set it aside Remove the filter base and set it aside Discard the used filter assembly in an appropriate receptacle To attach the new filter L Connect the vent line to the new filter assembly Twist to attach Wrap Teflon tape around the filter threads then connect the filter to the filter base Connect the sheath line to the filter assembly by squeezing the quick disconnect Attach the cytometer fluid line to the filter assembly via the quick disconnect Direct the vent line into a beaker and press the small button at the end of the vent fitting against the side of the beaker until a steady stream of fluid empties from the filter Tilt the filter and verify that no trapped air remains in the filter Repeat steps 5 and 6 as neces
69. n page 139 Two color red trigon configuration page 142 Two color UV laser configuration page 148 Four color blue laser configuration page 139 Two color red trigon configuration page 142 Two color violet laser configuration page 145 Five color blue laser configuration page 140 Three color red laser configuration page 143 Two color UV laser configuration page 148 Five color blue laser configuration page 140 Three color red laser configuration page 143 Three color violet laser configuration page 146 Five color blue laser configuration page 140 Three color red laser configuration page 143 Six color violet octagon page 147 Number of lasers Colors 3 8 8 10 11 14 Lasers 4 Blue 2 Red 2 UV 4 Blue 2 Red 2 Violet 5 Blue 3 Red 2 UV 5 Blue 3 Red 3 Violet 5 Blue 3 Red 6 Violet Chapter 9 Detector array configurations 135 See section Four color blue laser configuration page 139 Two color red trigon configuration page 142 Two color UV laser configuration page 148 Four color blue laser configuration page 139 Two color red trigon configuration page 142 Two color violet laser configuration page 145 Five color blue laser configuration page 140 Three color red laser configuration page 143 Two color UV laser configuration page 148 Five color blue laser configuration page 140 Three color red laser configuration page 143 Three color violet laser configuration page 14
70. nd the MyDataAnalysis global worksheet icon in the Browser Applying an analysis to normal worksheets Related topics Chapter 6 Recording and analyzing data 95 2 Right click its analysis and select Copy EE FourColorBeads HR 7 Cytometer Settings a Global Worksheets aE My Data Analysis EEE 3 Click the Worksheets View button on the Worksheet toolbar to switch to the normal worksheet view 4 Select Worksheet gt New Worksheet to create a new normal worksheet 5 Right click the Beads_001 tube icon in the Browser and select Paste ax FourColorBeads no TP Beads 00 H Beads_o02 a Shared View el Bl bi Cut Ctrl x Copy Chr Delete Delete The analysis objects from the MyDataAnalysis global worksheet are copied to the Beads_001_Analysis normal worksheet Double click the Beads_001 tube in the Browser to view the analysis You can apply the global worksheet analysis to multiple tubes on a single normal worksheet by selecting multiple tubes before pasting the analysis Ensure that you collapse all tube elements in the Browser before you paste them to multiple tubes e Analyzing data page 88 Need more information fast Information about this product is also available in your software s Help system You can keep the Help window open while you use the software or print the information directly from the window Internet access is not required to use the Help system Technical overvi
71. ng Place the new O ring into the retainer Make sure the O ring is seated properly in the bottom of the retainer Replace the outer sleeve in the retainer Re install the retainer and the outer sleeve Install a sample tube on the SIP to ensure that the outer sleeve has been properly installed If the sleeve hits the bottom of the tube loosen the retainer slightly and push the sleeve up as far as it will go Tighten the retainer Need more information fast Information about this product is also available in your software s Help system You can keep the Help window open while you use the software or print the information directly from the window Internet access is not required to use the Help system Optimizing cytometer settings This section includes the following topics Cytometer settings workflow page 58 Verifying the configuration and user preferences page 61 Running a performance check page 64 Setting up an experiment page 69 Creating application settings page 73 Recording compensation controls page 76 Calculating compensation page 80 58 BD LSRFortessa Cell Analyzer User s Guide Cytometer settings workflow About this topic When to optimize settings Manual compensation First time users Before you begin This topic describes how to optimize cytometer settings The optimization is performed using the Cytometer Setup and Tracking Application Settings and Compensation Setup
72. ng table can be used with this laser wavelength for cell surface marker live dead discrimination and cell cycle applications See Common violet laser configurations page 157 for common octagon and trigon configuration maps Fluorochromes V450 Pacific Blue AmCyan Alexa Fluor 430 V500 DAPI Qdot 525 Qdot 545 Qdot 565 Qdot 585 Qdot 605 Qdot 655 Qdot 700 Qdot 705 Qdot 800 BP filters 450 50 450 40 450 20 440 40 510 50 525 50 NA NA 560 20 585 15 605 12 605 40 655 8 NA NA NA 151 152 BD LSRFortessa Cell Analyzer User s Guide UV 355 nm 20 100 mW The fluorochromes listed in the following table can be used with this laser wavelength for the cell cycle application with the DAPI and Hoechst fluorochromes The wavelength is used for the Ca flux application with both Indo 1 fluorochromes See Common UV laser configurations page 159 for two common trigon configuration maps Fluorochromes BP filters DAPI Hoechst 440 40 450 20 450 40 450 50 Indo 1 Ca Bound 450 50 Indo 1 Ca unbound 530 30 Yellow Green 561 nm The fluorochromes listed in the following table can be used 50 mW with this laser wavelength for cell surface marker and fluorescent protein applications See Common yellow green laser configuration page 161 for a common octagon configuration map Fluorochromes BP filters PE DsRed 582 15 582 15 PE Texas Red mCherry 610 20 PE Cy5 660 20 670 30 PE Cy5 5 710 50
73. njection port and then screw the retainer into place and tighten to finger tight This will seat the Bal seal Install a sample tube on the SIP to ensure that the outer sleeve has been properly installed If the sleeve hits the bottom of the tube loosen the retainer slightly and push the sleeve up as far as it will go Tighten the retainer 54 BD LSRFortessa Cell Analyzer User s Guide Changing the sample tube O ring About this topic When to replace the O ring Caution A A Procedure This topic describes how to replace the sample tube O ring The sample tube O ring located within the retainer forms a seal that allows the droplet containment vacuum to function properly Replace the O ring when droplets form at the end of the sample injection tube while the vacuum is operating Caution Biohazard Cytometer hardware might be contaminated with biohazardous material Wear suitable protective clothing eyewear and gloves whenever cleaning the cytometer or replacing parts To change the O ring 1 Remove the outer sleeve from the sample injection tube by turning the retainer counter clockwise 2 Slide the outer sleeve from the retainer O ring Retainer Loan Chapter 4 Maintenance 55 Invert the outer droplet sleeve and allow the O ring to fall onto the benchtop If the O ring does not fall out initially hold the O ring with your free hand and slide the outer sleeve to remove the O ri
74. ometer page 43 Flushing the system page 44 Maintaining the waste management system page 46 Changing the sheath filter page 50 Changing the Bal seal page 52 Changing the sample tube O ring page 54 This topic describes how to perform the daily fluidics cleaning Cleaning the fluidics daily prevents the sample injection tube from becoming clogged and removes dyes that can remain in the tubing In addition to daily cleaning follow this procedure immediately after running viscous samples or nucleic acid dyes such as Hoechst DAPI propidium iodide PI acridine orange AO or thiazole orange TO 42 BD LSRFortessa Cell Analyzer User s Guide Procedure To clean the fluidics 1 2 FA y Press RUN and HI on the cytometer fluid control panel Install a tube containing 3 mL of a cleaning solution on the SIP with the support arm to the side vacuum on and let it run for 1 minute For the cleaning solution use BD FACSClean solution See Maintenance overview page 40 for other recommended cleaning solutions Move the tube support arm under the tube vacuum off and allow the cleaning solution to run for 5 minutes with the sample flow rate set to HI Repeat steps 2 and 3 with BD FACSRinse solution Repeat steps 2 and 3 with DI water Press the STNDBY button on the fluidics control panel Place a tube containing no more than 1 mL of DI water on the SIP A tube with 1 mL of DI water should
75. on its holder There are two types of optical filters in the BD LSRFortessa e Longpass dichroic filters LPs Transmit wavelengths that are longer than the specified value and reflect all light below the specified wavelength e Bandpass filters BPs Pass a narrow spectral band of light When dichroic filters are used as steering optics to direct different color light signals to different detectors they are called dichroic mirrors LP dichroic mirrors transmit longer wavelengths to one detector while reflecting shorter wavelengths to a different detector 20 BD LSRFortessa Cell Analyzer User s Guide Signal detectors The BD LSRFortessa cytometer octagon and trigon detector arrays use dichroic longpass mirrors on their inner rings and bandpass filters on their outer rings You can customize the arrays with other wavelengths of filters and mirrors Bandpass filters Longpass dichroic mirrors Light signals are generated as particles pass through the laser beam in a fluid stream When these optical signals reach a detector electrical pulses are created that are then processed by the electronics system There are two types of signal detectors in the BD LSRFortessa flow cytometer e Photomultiplier tubes PMTs Used to detect the weaker signals generated by side scatter and all fluorescence channels These signals are amplified by applying a voltage to the PMTs e Photodiodes Less sensitive to light signals t
76. on port SIP 15 fluidics controls cytometer 12 fluidics interconnect 29 fluorescence defined 101 emission 111 fluorescence signal none 120 fluorochromes compensation theory 108 emission spectra 102 measured 35 role in light emission 101 flushing the system 44 forward scatter FSC 100 FSC See forward scatter G gating data 88 global worksheets analyzing data 88 creating 83 previewing data 82 94 green 532 nm laser 153 162 H hazard symbol definitions 3 Help accessing 5 High Throughput Sampler HTS 16 hydrodynamic focusing 98 immunophenotyping analysis 88 experiment 83 hydrodynamic focusing 99 184 BD LSRFortessa Cell Analyzer User s Guide K keyboard conventions 4 knob SAMPLE FINE ADJ 13 L laser delay about 164 optimizing 165 lasers options 19 performance check 64 quality control QC particles 177 longpass LP filters defined 103 holder 34 theory 104 LP See longpass filters M maintenance Bal seal 52 battery change 47 battery test 46 cytometer shutdown 43 sample tube O ring 54 schedule 41 scheduled 44 sheath filter 50 system flush 44 waste management system 46 mirrors dichroic longpass LP filter 20 N notch filters 104 155 O octagon configuration maps 132 detector 10 location 36 shown 20 online Help 5 optic holder 35 optics components 18 configuration 61 62 dichroic mirrors 107 filters 19 36 103 location 11 optimization sample 58 ordering spare p
77. pter 8 Troubleshooting 115 Cytometer troubleshooting 0 000 eee eee e teenies 116 PleCtrOniCstrOuUblEShOOUNG 4 0 4 a tarinar PRs Badd r Dae Wek 127 Chapter 9 Detector array configurations 129 Figos nce SPECIE A vanced tease a alata eseok aoe hee ached peat eae ees 130 Ab utconfig ration maps 2 ds sean 6 at eR haeae BRA aa re Cee aes 132 About the Dase CONNGUTATION 6 224 55 5 c bh S Ae ee ADO DMS oS 133 Base configuration octagon and trigon maps 0 cee eee ee ee eens 138 Special Order configurations essees osen EON 4 Oho SOR SRE 149 Chapter 10 Manual settings 163 ADONCA OAC jacana hoa ya a aoe eee ee eee 164 Optimizing laser delay s c iw acon Oa ew ee Sods eee Re Od Ree Seed 165 Adjusting area scaling 3405463 haa i eke ee eh Se OTR AIA MORES EORSS 168 Chapter 11 Supplies and consumables 175 Ordering informa gt 240 aes enc ae e Sk he Bee a le wa Pe 176 Parcel desea c sa seetoces cbt E ae ale soacsiose a Shp a aso ae 177 PRCA EE ccs 2a E cend ese Wise or suerte dane SEE eee est Sh gee IG ee ee 178 PGUIPIMNeNE ctecwass oat Soothe h eet eet aee es eee aes ee ete hoes eee 179 Index 181 About this guide This section includes these topics e What this guide covers page 2 e Conventions used in this guide page 3 e About the BD LSRFortessa documentation page 5 e Technical assistance page 7 2 BD LSRFortessa Cell Analyzer User s Guide What this guide covers This guide describes the procedu
78. res necessary to operate and maintain your BD LSRFortessa cell analyzer Because many cytometer functions are controlled by BD FACSDiva software this guide also contains information about software features required for basic cytometer setup and operation This guide assumes you have a working knowledge of basic Microsoft Windows operation If you are not familiar with the Windows operating system see the documentation provided with your computer Chapter 1 About this guide 3 Conventions used in this guide About this topic This topic describes the conventions used throughout this guide Safety symbols These safety symbols are used in this guide and on safety labels to alert you to potential hazards Meaning Caution alert Identifies a hazard or unsafe practice that could result in data loss material damage minor injury severe injury or death Biological hazard Electrical hazard Laser hazard Hot surface High temperatures Burn hazard 4 BD LSRFortessa Cell Analyzer User s Guide Text and keyboard conventions Convention Notes Bold Italics Ctrl X Use A note describes important features or instructions Bold is used to indicate software elements such as dialogs fields and buttons Italics are used to highlight book titles Italics are also used in describing software to indicate specific text typed into a window or dialog The arrow is used to indicate menu selection p
79. romes A 750 780 60 APC Cy7 APC H7 B Blank 670 14 APC Chapter 9 Detector array configurations 143 Three color red laser The following map shows the three color configuration for configuration the 640 nm red laser PMT LP mirror BP filter Fluorochromes A 750 780 60 APC Cy7 APC H7 710 730 45 Alexa Fluor 700 C 670 14 APC 144 BD LSRFortessa Cell Analyzer User s Guide Four color red laser configuration the 640 nm red laser PMT LP mirror BP filter A 750 780 60 B 710 730 45 C 675 685 35 D 670 14 The following map shows the four color configuration for Fluorochromes APC Cy7 APC H7 Alexa Fluor 700 Alexa Fluor 680 APC Chapter 9 Detector array configurations 145 Two color violet laser The following map shows the two color configuration for configuration the 405 nm violet laser PMT LP mirror BP filter Fluorochromes A 475 525 50 AmCyan Qdot 525 BD Horizon V500 B Blank 450 50 Pacific Blue BD Horizon V450 Marina Blue Alexa Fluor 405 146 BD LSRFortessa Cell Analyzer User s Guide Three color violet laser The following map shows the three color configuration for configuration the 405 nm violet laser PMT LP mirror BP filter Fluorochromes A 595 605 12 Qdot 605 475 525 50 AmCyan V500 Qdot 525 C 450 50 Pacific Blue V450 Marina Blue Alexa Fluor 405 Six color violet octagon Chapter 9 Detector array configurat
80. rray configurations 159 Common UV laser The following maps show two common configurations for configurations the 355 nm UV laser Indo 1 violet OX Indo 1 blue 160 BD LSRFortessa Cell Analyzer User s Guide Indo 1 blue Hoechst Red Indo 1 violet Common yellow green laser configuration Chapter 9 Detector array configurations 161 The following map shows a common configuration for the 561 nm yellow green laser PE Cy5 5 PE Texas Red PE Cy5 Note that when you use a PE conjugate with a 561 nm laser you must replace the 585 42 bandpass filter BP with a 582 15 BP regardless of which laser blue green or yellow green is exciting the PE fluorochromes 162 BD LSRFortessa Cell Analyzer User s Guide Common green laser The following map shows a common configuration for the configuration 532 nm green laser PE Texas Red PE Cy5 10 Manual settings This section covers the following topics e About laser delay page 164 e Optimizing laser delay page 165 e Adjusting area scaling page 168 164 BD LSRFortessa Cell Analyzer User s Guide About laser delay About this topic About laser signal delay This topic describes how to manually set the laser delay if you are not using Cytometer Setup and Tracking Sample interrogation takes place within the cuvette flow cell Laser light is directed through a series of prisms that focus multiple la
81. ry in the BD FACSDiva Software Reference Manual The threshold is the level at which the system starts to measure signal pulses A threshold is defined for a specific detector signal The system continuously samples the digitized signal data and calculates pulse area height and width for all channels based on the time interval during which the threshold is exceeded Thresholds can also be set for more than one parameter and pulse measures are based on either of the following e Intervals during which ALL signals exceed their threshold value e Intervals during which ANY signal exceeds its threshold value 114 BD LSRFortessa Cell Analyzer User s Guide Laser controls Related topics Controls in the Laser tab of the Cytometer window are used to manually set the laser delay area scaling and window extension values These parameters are set by BD Biosciences service personnel during instrument installation and performance check and are updated each time you run a performance check If needed see Optimizing laser delay page 165 for instructions on manually adjusting laser delay settings Do not otherwise change the settings in the Laser tab unless instructed to do so by BD Biosciences Changing the settings affects your data Cytometer LSRFortessa 1 Threshold Laser Compensation Ratio IL foe am nl O a oF en Wino Extension e Running a performance check page 64 e Optimizing laser d
82. s are normalized to CS amp T settings In this case CS amp T will generate Qr and Br numbers that are not comparable from instrument to instrument Care should be taken when interpreting the CS amp T reported Qr values on Special Order BD LSRFortessa instruments Please see the latest published filter guides available on our website bdbiosciences com for more information The fluorochromes listed in the following table can be used with this laser wavelength for cell surface markers live dead discrimination and cell cycle applications See Common blue laser configurations page 154 for common octagon and trigon configuration maps Fluorochromes BP filters SSC 488 10 FITC Alexa Fluor 488 530 30 PE 575 25 5751 26 576 26 585 42 150 BD LSRFortessa Cell Analyzer User s Guide Fluorochromes BP filters PE Texas Red PI 610 20 616 23 PE Cy5 660 20 670 30 PerCP PerCP Cy5 5 PE Cy5 5 670 14 675 20 695 40 710 50 PE Cy7 780 60 Red 640 nm 40 mW The fluorochromes listed in the following table can be used with this laser wavelength for the cell surface marker application See Common red laser configurations page 156 for two common trigon configuration maps Fluorochromes BP filters APC Alexa Fluor 647 670 30 Alexa Fluor 680 685 35 Alexa Fluor 700 710 50 710 20 712 21 730 45 APC Cy7 APC H7 780 60 Violet 405 nm 20 100 mW Chapter 9 Detector array configurations The fluorochromes listed in the followi
83. s on page 28 124 BD LSRFortessa Cell Analyzer User s Guide Excessive amount of debris in display Possible causes Flow cell is dirty Air leak at sheath container Hypertonic buffers or fixative Possible causes Threshold level is too low Sheath filter is dirty Flow cell is dirty Dead cells or debris in sample Sample is contaminated Stock sheath fluid is contaminated Recommended solutions Flush the system See Flushing the system on page 44 Ensure that the sheath container lid is tight and all connectors are secure Replace the buffers or fixative Recommended solutions Increase the threshold level Replace the filter See Changing the sheath filter on page 50 Flush the system See Flushing the system on page 44 Examine the sample under a microscope Re stain the sample Ensure that the tube is clean Rinse the sheath container with DI water then fill the container with sheath fluid from another or new lot bulk container High CV or Poor QC results Possible causes Air bubble in sheath filter or flow cell Sample flow rate is set too high Air leak at sheath container Flow cell is dirty The dichroic mirrors are installed backwards Waste tank is pressurized Poor sample preparation Chapter 8 Troubleshooting 125 Recommended solutions e Purge the filter See Removing air bubbles on page 28 Prime the fluidics system See Priming the fluidics
84. sa Serial Number 1 Configurations Parameters Filters and Mirrors 6 Blue 4 Red 6 Yiolet 2 UY BD default Current Configuration Copy of 6 Blue 4 Red 6 iolet 2 UY H Base Configurations IB 6 Blue 4 Red 6 Violet 2 U BD default Blue Laser Red Laser Yiolet Laser UY Laser eS ae a 488nm 640nm 405nm 355nm Custom Configurations FSC all Blue Red Violet uv Window Extension us 10 00 138 BD LSRFortessa Cell Analyzer User s Guide Upgrade options Related topics The BD LSRFortessa cytometer optical configuration can be upgraded to use different special order wavelength options and a variety of detectors See Special Order configurations page 149 for a list of some common special order laser detector combinations available through the BD Special Order Research Program Many other special order configurations and options are also available e Verifying the configuration and user preferences page 61 e Base configuration octagon and trigon maps page 138 Base configuration octagon and trigon maps About this topic About the maps This section describes how filters and mirrors are arranged for standard trigon and octagon configurations The tables in this section show the detectors filters and mirrors used in the each configuration and recommended fluorochromes for each detector The word blank indicates that a blank optical holder should be used instea
85. sary to remove all trapped air 52 BD LSRFortessa Cell Analyzer User s Guide Changing the Bal seal About this topic When to change the Bal seal Procedure This topic describes how to replace the Bal seal The sample injection tube Bal seal is a ring that forms a seal with the sample tube and ensures proper tube pressurization Over time the Bal seal becomes worn or cracked and requires replacement Replacement is necessary if a proper seal is not formed when a sample tube is installed on the SIP Indications that a proper seal has not formed include e The tube will not stay on the SIP without the tube support arm e When the tube is installed and RUN is pressed on the cytometer the RUN button is orange not green To replace the Bal seal 1 Remove the outer sleeve from the sample injection tube by turning the retainer counter clockwise Slide the outer sleeve down and off of the sample injection tube Bal seal Retainer Outer sleeve Sample injection tube Work carefully The outer sleeve can fall off as you loosen the retainer Chapter 4 Maintenance 53 Remove the Bal seal by gripping it between your thumb and index finger and pulling down Install the new Bal seal spring side up Ensure that the sample tube O ring is still in place inside the retainer Re install the retainer and outer sleeve over the sample injection tube Push the outer sleeve all the way up into the sample i
86. sers on the event stream at different positions This allows optimal detection of fluorescent signals from each laser with minimal cross contamination from the other beams For example in a BD LSRFortessa four laser system the blue laser intercepts the stream first followed by the violet UV and red lasers Because the laser signals are spatially separated there is a slight delay between the detection of each laser s signal Event intercepts yellow green laser Event intercepts red laser Yellow green Event intercepts UV laser Event intercepts violet laser Red Event intercepts blue laser UV Violet Blue Time gt The yellow green laser is only available through the BD Special Order Research Program The laser delay setting in BD FACSDiva software is used to realign the signals so they can be measured and displayed on the same time scale Signals are aligned with respect to the blue laser so the blue laser will have a 0 delay value and the red laser will have the longest delay Chapter 10 Manual settings 165 Optimizing laser delay About this topic This topic describes how to optimize the laser delay using BD FACSDiva software Before you begin To optimize the delay for a given laser you must acquire events from a sample with a fluorescence signal excited by that laser Follow the procedures in Recording and analyzing data page 81 for sample optimization and acquiring data
87. ssociated with that configuration are updated to the latest run Using application settings provides a consistent and reproducible way to reuse cytometer settings for commonly used applications You can include area scaling adjustment in your application settings See Adjusting area scaling page 168 for more information Before you begin Perform the cytometer setup procedure and run a performance check for the configuration that will be used for the application Procedure To create application settings 1 Inthe open experiment right click Cytometer Settings in the Browser then select Application Settings gt Create Worksheet A second global worksheet is added with the plots created according to the selections in the Parameters tab 74 BD LSRFortessa Cell Analyzer User s Guide 1 napas H E a eO a 5 S JP ae E b aleh m a Global hesti P Globe Sheek Tonnen f k T j 7 All Events All Events All Events W m ag PerlP yo 5 4 Use the gray boxes and crosshairs to guide your optimization 2 Load the unstained control tube onto the cytometer 3 In the Cytometer window optimize the PMT voltages for the application Optimize the FSC and SSC voltages to place the population of interest on scale Optimize the FSC threshold value to eliminate debris without interfering with the population of interest If needed increase the fluorescence PMT voltages to place the negative population within
88. such as FITC have a smaller Stokes shift absorbing blue light 488 nm and emitting green light 530 nm 102 BD LSRFortessa Cell Analyzer User s Guide The following figure shows the emission spectra of some commonly used fluorochromes 100 AmCyan x m Alexa 700 c 2 uy at E 60 LLI T a JN 40 E 2 ZU 400 SOU 600 FOU Wavelength nm Actual emission intensity will depend on excitation wavelength See Fluorescence spectra page 130 for more information on excitation and emission of fluorochromes An interactive spectral viewer is also available at bdbiosciences com colors fluorescence_spectrum_viewer Optical filter theory Types of optical filters Chapter 7 Technical overview 103 Optical filters modify the spectral distribution of light scatter and fluorescence directed to the detectors When photons encounter an optical filter they are either transmitted absorbed or reflected Photons Barak absorbed aO transmitted reflected Even though an optical filter is rated at its 50 transmission point the filter passes lets through a minimal amount of light outside of this indicated rating The slope of an optical filter transmission curve indicates filter performance A relatively steep slope indicates a high performance high quality optical filter that provides deep attenuation of out of band wavelengths A less steep slope indicates that more light outside the rated bandwidth is being transmit
89. t the FSC and SSC voltages to place the particles on scale 200 240 e 1 000 10 Adjust the P1 gate around the population of interest 172 BD LSRFortessa Cell Analyzer User s Guide 11 Adjust the FSC area scaling a Click the Laser Tab in the Cytometer window Cytometer Z5A100002 LSRFortessa 71 Status Paramete 33 11 0 55 75 17 0 48 a Exch 10 00 F Fi b Adjust the FSC area scaling factor until the FSC A signal matches the FSC H signal e Increase the area scaling factor if the FSC A signal is lower than FSC H e Decrease the area scaling factor if the FSC A signal is higher than FSC H c View the result of your change in the histograms and statistics views 12 Adjust the blue laser area scaling factor until the FITC A signal matches the FITC H signal if needed 13 Unload the FITC positive control tube then load the APC positive control tube Chapter 10 Manual settings 173 14 Adjust the red laser area scaling factor until the APC A signal matches the APC H signal if needed Si SEEN 001 Tube 00i pecinen O01 Tube 001 200 Tube Mame Tube_O01 Record Date Det 1 2009 1057 48 AM FSC FS C H Population Mean hiean Pi 68 989 68 339 Need more information fast Information about this product is also available in your software s Help system You can keep the Help window open while you use the software or print the information directly from the window
90. ted There are four types of filters e Longpass LP filters Transmit wavelengths that are longer than the specified value e Shortpass SP filters Transmit wavelengths that are shorter than the specified value This type of filter is not recommended but can be used in some custom configurations See Shortpass SP filters on page 105 e Bandpass BP filters Pass a narrow spectral band of light by combining the characteristics of shortpass filters longpass filters and absorbing layers 104 BD LSRFortessa Cell Analyzer User s Guide Longpass LP filters e Notch filters Pass all frequencies except those in a stop band centered on a center frequency They are the opposite of bandpass filters The LSRFortessa uses LP filters and BP filters Notch filters are sometimes used in special order BD LSRFortessa instruments LP filters pass wavelengths longer than the filter rating For example a 500 LP filter permits wavelengths 500 nm or longer to pass through it and either absorbs or reflects wavelengths shorter than 500 nm Longpass 480 520 460 500 540 Transmission 450 500 550 Wavelength nm Shortpass SP filters Chapter 7 Technical overview 105 An SP filter has the opposite properties of an LP filter An SP filter passes light with a shorter wavelength than the filter rating For example a 500 SP filter passes wavelengths of 500 nm or shorter and reflects or absorbs wavelengths longer than
91. ter Caution Pressurized contents might spray Use appropriate cautionary measures 1 Slowly loosen the waste tank cap to relieve the pressure in the waste tank 2 Remove the waste vent filter by rotating counter clockwise 3 Install a new waste vent filter Replace the sample tube Connect the waste tubing line to the waste cap 120 BD LSRFortessa Cell Analyzer User s Guide No fluorescence signal Possible causes Sheath container is not pressurized Bal seal is worn Air leak at sheath container Sheath container is empty Air in sheath filter Possible causes Incorrect fluorochrome assignment Wrong filter is installed Laser is not functioning Recommended solutions e Ensure that the sheath container lid and all connectors are securely seated e Inspect the sheath container O ring inside the lid and replace it if necessary Replace the Bal seal See Changing the Bal seal on page 52 Ensure that the sheath container lid and all connectors are securely seated Fill the sheath container Purge the filter See Removing air bubbles on page 28 Recommended solutions Make sure that the cytometer configuration in the software matches the optical filters in the cytometer and the configuration is as expected Make sure the appropriate filter is installed for each fluorochrome See Changing optical filters and mirrors on page 36 Call your BD service representative
92. ter 10 components shown 11 configuration 35 61 disconnect error 127 door and drawer 36 setup 58 starting 24 troubleshooting 116 D DAPI cleaning after using 41 data analyzing 82 88 gating 88 recording 82 85 debris excessive 124 default configuration 133 delay laser 164 detector arrays 18 detectors 20 dichroic mirrors slots 34 theory 107 digital data 10 digital electronics 113 Diva software See software DNA flow rate for analysis 99 droplet containment system 16 E electronics digital 113 laser controls 114 pulse 111 pulse measurements 112 threshold 113 troubleshooting 127 emission duration 111 event rate erratic 123 high 121 low 122 zero 117 119 excessive debris 124 excitation wavelength 101 experiments creating 69 immunophenotyping 83 sample optimization 69 specifying parameters 70 F FACSFlow sheath fluid 178 FACSFlow solution 27 Falcon tubes 117 179 filters optical bandpass BP 20 106 changing 36 dichroic See dichroic mirrors longpass LP 103 104 notch 104 155 shortpass SP 103 105 slots 34 theory 103 filters sheath attaching new 51 replacing 50 FITC and Stokes shift 101 fixed alignment lasers 10 flow cell draining 33 fluidics 98 flow rate control buttons 13 fluid control buttons PRIME 14 RUN 14 STNDBY 14 fluidics description 13 98 flow rate control 13 flushing system 44 priming 33 Index 183 removing air bubbles 28 run mode 14 sample injecti
93. the fluidics page 33 About the optical filters and mirrors page 34 Changing optical filters and mirrors page 36 Custom configurations and baselines page 37 24 BD LSRFortessa Cell Analyzer User s Guide Starting the cytometer and computer About this topic Procedure This topic describes how to start the cytometer and turn on the computer To start the cytometer L Turn on the power to the flow cytometer Note If your system is using the BD FACSFlow supply system make sure that the BD FACSFlow supply system is powered on before the cytometer Allow 30 minutes for the optical system temperature to stabilize Caution Failure to warm up and stabilize the instrument could affect sample data Turn on the computer and log in to Windows Note You can turn on the power to the flow cytometer and the workstation in any order Start BD FACSDiva software by double clicking the shortcut on the desktop and log in to the software Chapter 3 Cytometer setup 25 Check the Cytometer window in BD FACSDiva software to ensure that the cytometer is connected to the workstation The cytometer connects automatically While connecting the message Cytometer Connecting is displayed in the status area of the Cytometer window When connection completes the message changes to Cytometer Connected Cytometer LSRFortessa 1 If the message Cytometer Disconnected appears see Electronics troubleshooting page
94. tor Cytometer Settings Cytometer Settings Parameters Threshold Ratio Compensatic Parameter We b Select the specific parameter from the drop down menu Your selection appears as the selected parameter Inspector Cytometer Settings Cytometer Settings Parameters Threshold Ratio Cor Parameter e FS e FITE ka e Alexa Fluor 488 pe c For this example select FITC from the menu 72 BD LSRFortessa Cell Analyzer User s Guide 3 Delete any unnecessary parameters a Click the selection button to the left of the parameter name to select the parameter Inspector Cytometer Settings Cytometer Settings Parameters Threshold Ratio Compensation Parameter Voltage Log FSC 250 55C 300 FITC 500 PE PerCP Cy5 5 y2E Cy K 500 Pacific Blue gt poBooooe e AmCyan Indo 1 Violet e Indo 1 Blue b ae b OOO b Click Delete The parameter is deleted Chapter 5 Optimizing cytometer settings 73 Creating application settings About this topic This topic describes how to create application settings About application Application settings are associated with a cytometer settings configuration and include the parameters for the application area scaling values PMT voltages and threshold values but not compensation Each time a performance check is run for a configuration the application settings a
95. trum_viewer The BD LSRFortessa cytometer is designed specifically for multicolor research There are many options for dyes reagents and system configurations When choosing a configuration or panel it is important to remember that final results depend on the excitation and emission spectra of the individual dye the number of fluorescently labeled binding sites on the cell as well as spectral overlap and spillover to other PMTs For more information about designing multicolor panels see Selecting Reagents for Multicolor Flow Cytometry Part No 23 9538 02 Chapter 9 Detector array configurations 131 Example laser and dye The following figure shows the emission spectra of some interactions common dyes based on laser excitation In many cases a given dye can be excited by multiple laser wavelengths yielding different emission intensities UV 355 nm Hioechsta258 Violet 405 nm Blue 488 nm Yellow Green 561 nm Available only on Special Order Research Products Red 640 nm 132 BD LSRFortessa Cell Analyzer User s Guide About configuration maps About this topic Filter and mirror arrangement This topic describes the filter and mirror arrangements in the detector arrays The filters are arranged in the detector array to steer progressively shorter wavelengths of light to the next PMT in the array The longest wavelength should be in the A position and the shortest wavelengt
96. ulation Singlets Chapter 6 Recording and analyzing data 89 Select all plots except the FSC vs SSC plot and use the Plot tab in the Inspector to specify to show only the singlet population Inspector Dot Plot Flot Tube 4 Parameter Y Parameter Biexponential Display Axis Axis Plot Elements Grid Plot Cutline Tick Labels Background Color Grid Outline Tick Marks Full C Half Population all Events WW singlets 90 BD LSRFortessa Cell Analyzer User s Guide 8 Select all plots and click the Title tab in the Inspector Select the Tube and Populations checkboxes to display their names in plot titles Inspector Dot Plot Custom Titles Title Font Italic _ Bold 9 On all fluorescence plots Make all plots biexponential Select all fluorescence plots and select the X Axis and Y Axis checkboxes in the Plot tab of the Inspector Inspector Dot Plot Plat Title Labels Acquisition Dot Plot Tube eads Specimen _001 Beads _001 Parameter FSC A w Parameter 55 A Cytometer Setup AS pomsctial Display x Axis Y Axis Inthe FITC vs PE plot draw a gate around the FITC positive population Name the population FITC positive in the population hierarchy Chapter 6 Recording and analyzing data 91 Inthe FITC vs PE plot draw a gate around the PE positive population Name the
97. used laser beam laser light is scattered in all directions Light that scatters axial to the laser beam is called forward scatter FSC and light that scatters perpendicular to the laser beam is called side scatter SSC Side scatter SSC Light source Forward scatter FSC Fluorescence Chapter 7 Technical overview 101 FSC and SSC are related to certain physical properties of cells e FSC Indicates relative differences in the size of the cells or particles Larger cells scatter more light and therefore they are higher in FSC e SSC Indicates relative differences in the internal complexity or granularity of the cells or particles More granular cells deflect more light than less granular cells and therefore are higher in SSC When cells or particles stained with fluorochrome conjugated antibodies or other dyes pass through a laser beam the dyes can absorb photons energy and be promoted to an excited electronic state In returning to their ground state the dyes release energy most of which is emitted as light This light emission is known as fluorescence Fluorescence is always a longer wavelength lower energy photon than the excitation wavelength The difference between the excitation wavelength and the emission wavelength is known as the Stokes shift Some fluorescent compounds such as PerCP exhibit a large Stokes shift absorbing blue light 488 nm and emitting red light 675 nm while other fluorochromes
98. valid baseline defined If not see the BD Cytometer Setup and Tracking Application Guide for more information on defining a baseline 5 Prepare the CS amp T beads according to the technical data sheet provided with the beads or available on the BD Biosciences website bdbiosciences com 6 Install the bead tube onto the SIP 7 Inthe Setup Control window select Check Performance from the Characterize menu Setup Control Research Use Only Load a tube with beads and click Run button bo start setup Characterize Check Performance gt Pun E Abort 10 11 12 Chapter 5 Optimizing cytometer settings 67 Click Run Ensure that the SAMPLE FINE ADJ knob is set to the midpoint and press RUN and LO Plots appear under the Setup tab and the performance check is run The performance check takes approximately 5 minutes to complete Once the performance check is complete click View Report Verify that the cytometer performance passed In the Setup tab the cytometer performance results should have a green checkbox displayed and the word Passed next to it EJ Cylomele Baretne January 18 2010 1 26 AM Citomeler Peformance January 20 2010 03203 FM gJ Comella Peleemance Assiz Passed If any parameters did not pass see the BD Cytometer Setup and Tracking Application Guide for troubleshooting information Select File gt Exit to close the CSXT window and return to the BD FACSDiva interface
99. vely shorter wavelengths of light to the next PMT in the array starting at PMT A Optic holders filters and mirrors Base configurations BD FACSDiva cytometer configuration Related topics Chapter 3 Cytometer setup 35 Optic holders house filters and mirrors Your cytometer includes several blank empty optic holders Note To ensure data integrity do not leave any slots empty in a detector array when you are using the associated laser Always use a blank optic holder Each BD LSRFortessa cytometer has a base cytometer configuration that corresponds to the layout of the installed lasers and optics in your cytometer This base configuration is set by your field service engineer Before you acquire data using BD FACSDiva software you must specify a cytometer configuration The cytometer configuration defines which filters and mirrors are installed at each detector BD FACSDiva software provides a BD base configuration for your BD LSRFortessa cytometer Select Cytometer gt View Configuration to create modify or delete custom cytometer configurations See the Cytometer and Acquisition Controls chapter of the BD FACSDiva Software Reference Manual for details e Changing optical filters and mirrors page 36 e About the base configuration page 133 e Special Order configurations page 149 36 BD LSRFortessa Cell Analyzer User s Guide Changing optical filters and mirrors About this topic A A Procedur
100. workspace 1 Using the Browser toolbar create a new specimen in MyExperiment and rename it FourColorBeads Create two tubes for the FourColorBeads specimen Rename the tubes Beads_001 and Beads_002 Expand the Global Worksheets folder in MyExperiment to access the default global worksheet and rename the worksheet MyData 84 BD LSRFortessa Cell Analyzer User s Guide Applying saved application settings to a new experiment Related topics On the MyData worksheet create the following plots for previewing the data FSC vs SSC FITC vs PE FITC vs PerCP Cy5 5 FITC vs APC When applications settings are applied to an experiment the cytometer settings are updated with the parameters included in the application settings optimized PMT voltages threshold settings area scaling factors and window extension values To apply saved application settings to your experiment L Right click the experiment level Cytometer Settings and select Application Settings gt Apply In the Application Settings catalog select the application settings file you saved previously and click Apply If the parameters are not the same a mismatch dialog opens Click Overwrite to update all settings Click Apply to change only the common parameters For more information see the BD FACSDiva Software Reference Manual The cytometer settings are renamed application settings and the cytometer settings icon in th
101. ys Caution Do not place any objects on top of the instrument Blocking the ventilation may cause the instrument to overheat j Caution Electrical Hazard Do not place liquids on top of the A AN instrument Any spill of liquid into the ventilation openings could cause electrical shock or damage to the instrument 12 BD LSRFortessa Cell Analyzer User s Guide Power switch The power switch is located on the right side of the instrument Control panel The control panel contains the following fluidics controls e Sample flow rate control buttons e Fluid control buttons e Sample fine adjust knob Sample flow rate control buttons Fluid control buttons Related topics e Fluidics page 13 e Optics page 18 Fluidics About this topic Purpose Sample flow rate control Chapter 2 Introduction 13 This topic describes the fluidics system The purpose of the fluidics system is to carry the sample out of the sample tube and into the sensing region of the flow cell Cells are carried in the sample core stream in single file and measured individually Three flow rate control buttons LO MED and HI set the sample flow rate through the flow cell The SAMPLE FINE ADJ knob allows you to adjust the rate to intermediate levels When the SAMPLE FINE ADJ knob is at its midpoint the sample flow rates at the LO MED and HI settings are approximately 12 35 and 60 pL min of sample respectively The knob turns five
102. ytometer settings should not require adjustment other than the changing of FSC and SSC voltages to place the beads on scale Adjust the P1 gate to surround only the singlets Right click the P1 gate and select Apply to All Compensation Controls The P1 gate on each stained control worksheet is updated with your changes Click Record Data When recording is finished remove the unstained control tube from the cytometer Click Next Tube Caution Do not change the PMT voltages after the first compensation control has been recorded In order to calculate compensation all controls must be recorded with the same PMT voltage settings If you need to adjust the PMT voltage for a subsequent compensation control you must record all compensation controls again Install the next tube onto the cytometer and repeat steps 8 through 10 until data for all stained control tubes has been recorded Double click the first stained control tube to display the corresponding worksheet Chapter 5 Optimizing cytometer settings 79 13 Verify that the snap to interval gate encompasses the positive population FITC Stained Control FITC Stained Control 40 Count i00 200 300 o a M a 2 HoN Ia L 17 on 2 oS we 150 700 2450 Foca te 1 000 14 Repeat steps 12 and 13 for the remaining compensation tubes Next step After you have recorded data for each single stained control calculate compensation as d

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