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FACSCalibur System User's Guide

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1. Figure 4 7 Time Delay Calibration document See Figure 4 8 13 14 FACSCalibur System User s Guide Secondary Param O O O O O S Figure 4 8 Slider pop up Threshold window Install a tube of APC beads on the SIP Note the current FSC amp gain value in the Detectors Amps window before you make the adjustment in step 14 You will need to return to this current setting after performing Time Delay Calibration Adjust the FSC amp gain to place the mean peak on the FSC histogram to Channel 400 5 Make sure the event rate is above 400 events second If the event rate is too low add more beads to the tube 67 Chapter 4 FL4 Option l 5 Choose Log as the Mode for FL4 Param Detector Yoltage Amp Gain Mode P2 SSC 350 a Y P3 FL1 550 3 1 800 it P4 FL2 600 Loaf P5 FL3 650 oof FLI A FLEW P7 FL4 800 e Log EJ Four Color DDM Param l Adjust the FL4 PMT voltage to place the mean peak in the FL4 histogram to Channel 800 5 l 7 Choose Time Delay Calibration from the Cytometer menu The Time Delay Calibration dialog box appears Time Delay Calibration Follow the calibration procedure in the Fatstaliaer User Gade 68 18 19 20 21 FACSCalibur System User s Guide Click Calibrate to begin the process The cursor idles for a couple of seconds while calibration takes place A beep sounds if the calibration is successful and the window disappears automatically
2. 20 0 eee eee eee 169 IMEX 4 4 5 5S oie aa na ea eas oo E eae wee oe ean eae ad 175 tii lv FACSCalibur System User s Guide Preface FACSCalibur is the Becton Dickinson Immunocytometry Systems BDIS modular benchtop flow cytometer designed for applications ranging from routine clinical to advanced research This modular system features advanced capabilities such as the Sorting and FL4 options in an easy to use system Integral to the FACSCalibur system is the FACStation Data Management system featuring a Macintosh computer and CELLQuest software a general purpose acquisition and analysis software program designed specifically for BDIS flow cytometers FACSComp instrument setup software is also included with the system Use FACSComp for daily FACSCalibur system quality control and setup Preface How to Use This Guide This user s guide contains the instructions necessary to operate and maintain your FACSCalibur flow cytometer The information is presented in easy to follow steps in boldface type followed by additional information that provides more detail Because many FACSCalibur functions are controlled by CELLQuest software you will also find the basic software information necessary for instrument setup If you are not familiar with the Macintosh computer or with CELLQuest software refer to the appropriate Macintosh user s guide provided by Apple Computer Inc and the CELL Quest Software User s G
3. _ NOTE If calibration is not successful the dialog box disappears and an error message dialog appears Click OK to remove the error dialog box and see Chapter 8 Troubleshooting Return the FSC threshold to 52 and the FSC amp gain values to their previous settings Choose Close from the File menu to remove the Time Delay Calibration Experiment document Remove the tube of APC beads from the SIP 69 Chapter 4 FL4 Option Setting Up the FL4 Parameter 2 2 Create a FL3 vs FL4 acquisition dot plot See Section 3 1 or refer to the CELL Quest Software Users Guide for instructions on creating dot plots 2 3 Place quadrants on the FL3 vs FL4 plot Use the Quadrant Marker tool to place markers as they appear in Figure 4 9 Quadrant Marker tool apg 1000 o fo wo o fo Tr o fo fam 200 400 800 6800 1000 FL3 H Figure 4 9 Quadrant markers placed 70 FACSCalibur System User s Guide 2 4 Install a tube of APC beads on the SIP 2 5 If necessary adjust the FL4 PMT to place the bead population in the target channel recommended in the APC Beads package insert 800 61000 FLAH fm wo o T o cm 200 400 B00 B00 1000 Figure 4 10 Adjusted FL4 voltage There is little or no FL4 autofluorescence from unlabeled beads Because of this you should use APC beads to adjust the FL4 PMT Unlabeled CaliBRITE beads are chosen to have fluorescence similar to the autofluorescence of l
4. Waste tubing carries waste fluid to waste reservoir Waste air vent tubing allows air to escape from waste reservoir as it fills Fluid detection probe cables connects fluid level sensors in sheath and waste reservoirs to system electronics Vent valve toggle switch telieves the sheath reservoir of air pressure when set in the direction of the arrow thus allowing for the removal of the reservoir when refilling 15 Chapter 2 Getting Started Filling the Sheath Reservoir l Slide out the fluidics drawer If the FACSCalibur instrument is powered on push the STNDBY button and flip the vent valve toggle switch located between the reservoirs This switch relieves the air pressure in the sheath reservoir 2 Slide the metal bracket away from you and lift up to remove it 3 Disconnect the sheath tubing white and the air supply tubing blue from the FACSCalibur instrument Squeeze the metal clip on the quick disconnects and pull each connector from the fitting 4 Disconnect the sheath fluid detection probe cable Squeeze the tabs at the sides of the connector and pull FACSCalibur System User s Guide Remove the sheath reservoir Unscrew the cap assembly from the reservoir and set the assembly aside Fill the reservoir with sheath fluid to 3 4 capacity See Appendix A Consumables and Service Information for the recommended sheath fluid A CAUTION Avoid filling the sheath reservoir to its maximum capacity
5. 31 Chapter 3 Instrument Setup for Acquisition of Samples 52 2 Click Save A standard directory dialog box appears Macintosh APPLICATIONS pippi SYSTEM FOLDER C untitled folder Save as Enter a name in the Save as field and choose a storage location for the file from the pop up menu These settings may be restored to the cytometer in the future 4 Click Save The Instrument Settings window appears Click Done to remove the window CHAPTER 4 FL4 Option CHAPTER 4 Summary FL4 optics time delay electronics dual threshold setting up the FACSCalibur instrument for 4 color analysis time delay calibration 4 1 FACSCalibur System User s Guide The FACSCalibur FL4 option increases multicolor analysis capability with the addition of a second laser and a PMT to detect the fourth fluorescence parameter The FL4 option includes modifications to the excitation and collection optics and electronics This chapter reviews these modifications and demonstrates how to set up the FACSCalibur instrument for 4 color acquisition using CaliBRITE beads Optics The standard laser included in the FACSCalibur system is a 15mW 488 nm air cooled argon ion laser The FL4 option provides a second laser an 635 nm red diode laser Multi laser cytometers from BDIS incorporate spatially separated beam geometry the first and second lasers are focused at different locations along the sample stream The
6. The sample injection tube Bal seal is a Teflon ring that forms a seal with the sample tube and creates proper tube pressurization Over time this 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 l Remove the outer droplet sleeve from the sample injection tube by turning the retainer counterclockwise Figure 7 4 Work carefully since the outer sleeve may fall out as you loosen the retainer Remove the Bal seal by gripping it between your thumb and index finger and pulling Figure 7 5 Bal seal Figure 7 4 Removing outer sleeve Figure 7 6 Removing Bal seal 3 Install the new Bal seal spring side up FACSCalibur System User s Guide 145 Chapter 7 Cleaning and Maintenance 146 A Reinstall the retainer and outer sleeve over the sample injection tube Tighten the retainer by turning it clockwise Gently push the seal in place to seat it If the seal does not remain in position when you let go hold it with one hand while you reinstall the retainer The seal will seat as you screw on the retainer Tighten the retainer just enough to hold it in place Slide the outer sleeve over the sample injection tube and into the opening of the retainer Continue tightening the retainer Install a sample tube on the SIP to ensure the outer sleeve has been properly installed If the sleeve hits the bottom
7. Cause Recovery mode used to sort Solution Use Single Cell or Exclusion mode to achieve high purity Cause Flow rate set to MED or HI Solution Set flow rate to LO when sorting SYMPTOM Cause Solution Cause Solution Cause Solution Cause Solution FACSCalibur System User s Guide Air bubbles in sample when reanalyzed Gently mix the sample to avoid air bubble formation before reanalyzing Air bubbles in flow cell Press PRIME to drain and fill flow cell See Section 2 2 3 step 2 Air bubbles in the sheath filter Push the roller in the pinchcock forward to purge air from the filter Threshold too high Reduce threshold as low as possible PRECISION High CV Cause Solution Cause Solution Cause Solution Cause Solution Air bubbles in flow cell Press PRIME to drain and fill flow cell See Section 7 2 Sample flow rate set to HI Set sample flow rate to LO or MED Improper sheath pressure Ensure sheath reservoir cap is tight and all connectors are secure Check sheath voltage in Status window Check also for a cracked sheath reservoir Flow cell dirty Perform monthly cleaning procedure See Section 7 1 2 159 Chapter 8 Troubleshooting SYMPTOM 160 Cause Solution Cause Solution Cause Solution Cause Solution Sample preparation technique questionable Seek advice on sample preparation techniques Air bubb
8. Experiment documents software documents containing any information entered such as plot formats page layout statistical markers and acquisition setup options CHAPTER 3 Instrument Setup for Acquisition of Samples CHAPTER 3 Summary E accessing instrument controls E optimizing instrument settings saving instrument settings 3 1 FACSCalibur System User s Guide Accessing Instrument Controls in CELLQuest The FACStation computer controls the FACSCalibur instrument electronics so any adjustments made to the instrument s detectors or amplifiers are made through CELLQuest software Turn on the FACSCalibur instrument before turning on the computer to ensure proper initialization between the cytometer and the computer In order to easily analyze flow cytometric data it is necessary to adjust the cytometer to optimally view the data prior to acquisition In this chapter you will learn how to access and adjust the cytometer settings in CELLQuest software You will then practice adjusting the instrument settings using CaliBRITE beads All adjustments to the FACSCalibur can be made through the Cytometer menu in CELLQuest software Cytometer Detectors Amps Threshold Compensation Status Pippe Ebath dehy U dEEEERE GEG Eddy Detectors Amps The Detectors Amps window Figure 3 1 allows you to adjust the detectors and amplifiers so that the signals appear appropriately on the data plots The light signals are
9. FL1 compensation value to rid the FL2 detector of FITC fluorescence overlap Notice the FITC labeled beads move toward the x axis FL1 Continue to adjust until the entire population is below the horizontal marker line Beads 005 Beads 008 Figure 3 13a Unadjusted compensation Figure 3 13b Adjusted compensation FACSCalibur System User s Guide FITC has a characteristic emission spectrum with a constant relationship between the amount of light in FL1 and FL2 The compensation value reflects this constant relationship Even though the relative light emission of FITC in each channel is always the same you will change the relative signal strengths if you change the PMT voltages thus affecting compensation This is why you adjust the PMT voltages before you adjust compensation OPTIONAL EXERCISE To further understand this concept do the following Increase the FL2 PMT by 20 volts Observe how FITC becomes undercompensated Make sure you return the FL2 PMT to its previous setting before you proceed 2 5 Adjust the FLI FL2 compensation Increase the FLI FL2 compensation value to rid the FL1 detector of PE fluorescence overlap Notice the PE labeled beads move toward the y axis FL2 Continue to adjust until the entire population is to the left of the vertical marker line Figure 3 14 2 6 Adjust the FL3 FL2 compensation while viewing the FL2 vs FL3 plot Increase the FL3 FL2 compensation value to rid the FL3 det
10. Fill the waste reservoir to 10 capacity 400 mL with undiluted household bleach Replace the cap assembly on the reservoir If necessary adjust the cap assembly on the reservoir so the tubing is not pinched or twisted and reaches the connectors on the connector panel FACSCalibur System User s Guide 9 Install the reservoir l Snap the waste and air vent tubing into their color coded fittings by pushing firmly until you hear a click l l Reconnect the waste fluid detection probe cable Priming the Fluidics Check the sheath filter for trapped air bubbles Vent the air from the filter if necessary Trapped bubbles can occasionally dislodge and pass through the flow cell resulting in inaccurate data If bubbles are visible gently tap the filter body with your fingers to dislodge the bubbles and force them to the top Push the roller in the pinchcock forward to allow the pressurized sheath fluid to force the air bubbles into the waste reservoir Return the pinchcock to the closed position To remove stubborn bubbles squeeze the metal clip and pull the sheath filter from the lower quick disconnect port Lift the filter up and firmly tap the filter body to dislodge the bubbles Reconnect the filter to its lower quick disconnect port Push the roller in the pinchcock forward to allow the ees 35 21 Chapter 2 Getting Started 22 pressurized sheath filter to force air bubbles into the waste reservoir Return the pin
11. Hone Figure 3 7 Threshold window Notice that forward scatter is selected as the threshold parameter in the Threshold window Choose Compensation from the Cytometer menu The Compensation window appears Use this window to adjust for overlapping emissions of the various fluorochromes in each sample When compensation is correct each fluorochrome is represented by one axis of the plot This simplifies data interpretation o o FL o o FLI o o FL3 o o FL a atlas FLA ngi a aft a FL3 inet 41 Chapter 3 Instrument Setup for Acquisition of Samples 42 l l Introduce the tube of unlabeled CaliBRITE beads on the SIP Swing the arm out and remove the tube of water Install the sample tube so the top of the tube is snug with the Bal seal Swing the arm into place under the tube Make sure there is a few millimeters of clearance between the bottom of the tube and the tube stop See Figure 2 3 in Chapter 2 l 2 Choose Counters from the Acquire menu The Counters window appears Use this window to view the Events Second rate before clicking Acquire There is a brief period after installing a tube when the Events Second rate may be erratic It is important to wait for it to stabilize it will take approximately 5 seconds Total Events O Events Second 0 Elapsed Time l 3 Push the RUN button on the FACSCalibur flow cytometer Make sure the button turns green in color If it does no
12. The Sort Setup window allows you to control all sorting options by selecting the gate to be used for sorting the number of cells to be sorted and the sort mode Install the sample tube on the SIP quickly center the tube support arm under the tube and press the RUN fluid control button Make sure the LO flow rate is selected Chapter 5 Sorting Option 92 2 Click Acquire in the Acquisition Control window As the sample is sorted a static like sound indicates sorting is taking place 3 Sorting stops when the first of four conditions is met See Section 5 7 Ending Sorting for details 5 7 Ending Sorting There are four ways to end sorting When any two or more of these parameters are used simultaneously sorting stops when the first parameter is reached l Manually lIn the Acquisition Control window click Pause and then Abort By Acquisition count lIn the Acquisition amp Storage window set Collection Criteria to Event Count and enter the number of events to acquire sorting stops when this number is reached if you are acquiring data to a file By Sort Count In the Sort Setup window set Sort Count to the number of cells to be sorted sorting stops when this number is reached By Time In the Acquisition amp Storage window set Collection Criteria to Event Count or Time and enter the sort time sorting stops when the event count or time is reached if you are acquiring data to a file FACSCalibu
13. When the reservoir is filled beyond the recommended level fluid may backflow into the air supply tubing preventing proper pressurization and potentially damaging the instrument Replace and tighten the cap assembly on the reservoir A securely tightened cap prevents air from leaking from the reservoir when the system is pressurized If necessary adjust the cap assembly so the tubing is not pinched or twisted and reaches the connectors on the connector panel Failure to securely tighten the cap could result in lack of sample flow and poor sorting pulse processing or FL4 results Chapter 2 Getting Started 9 10 11 12 13 Install the reservoir Replace the bracket Lower the bracket over the reservoir with the ball valve tab toward the middle of the drawer Pull the bracket toward you to lock it in place When correctly in place the ball valve tab depresses the ball valve to achieve accurate pressurization of the sheath reservoir Snap the fluid and air supply tubing into their color coded fittings by pushing firmly until you hear a click Reconnect the sheath fluid detection probe cable Remember to set the vent valve toggle switch back to its original position to pressurize the reservoir Check to see that the sheath reservoir fits snugly beneath the bracket The reservoir does not move when the system is fully pressurized When the FACSCalibur flow cytometer is in standby mode the sheath voltage displayed i
14. Dickinson Immunocytometry Systems Customer Support Center at 800 448 2347 BDIS Customers outside the US contact your local Becton Dickinson representative or distributor viii FACSCalibur System User s Guide Safety and Limitations Please read the following warnings and safety limitations This information should be kept available for future reference and for new users BDIS strongly recommends the FACSCalibur flow cytometer be operated only as directed in this user s guide the CELL Quest Software Users Guide and any accompanying manual for accessories and optional equipment Electrical Safety e For protection against shock equipment should be connected to an approved power source If an ungrounded receptacle is encountered have a qualified electrician replace it with a properly grounded receptacle in accordance with the Electrical Code e For installation outside the US a power transformer conditioner is necessary to accommodate 100 V 10 220 V 10 240 V 10 50 60 Hz 2 Hz 20 A Please contact your local Becton Dickinson office for further information e Do not under any circumstances remove the grounding prong from the power plug Do not use extension cords e Do not perform any servicing except as specifically stated in this user s guide az 1X Safety and Limitations Laser Safety The FACSCalibur instrument is a Class I laser product The laser is fully contained within the instrument str
15. FSC and one FL4 The Time Delay Calibration electronics will use FSC signals and FL4 signals To perform the calibration you will need to adjust the FSC and FL4 instrument settings Choose Threshold from the Cytometer menu The Threshold window appears 65 Chapter 4 FL4 Option 2 Adjust the FSC threshold to 200 using the slider pop up 66 Time Delay Calibration DICKINSON Time Delay Calibration Before proceeding make sure you have cormected to the cytometer selected Connect to Cytometer in the Acquire menu turned on the second laser selected the 4Colox checkbox in the Detectors Amp window selected Logas the mode for FLA in the Detectors Amp window set FL3 FL4 and FL4 FL3 compensations to 0 0 ition Histogram Plot Histogram Statistics File Acquisition Date Gate No Gate X Parameter FSC H Log Marker Mean All aut Mi 600 1000 STEP 1 Select FSC as the threshold parameter and set the threshold value to 200 STEP2 Adjust the FSC Amp Gain until the mean of the peakis at channel 40045 sition Histogram Plot Histogram Statistics File Acquisition Date Gate No Gate X Parameter FL24 Linear Marker Mean All i Mi 400 600 1000 FL2 STEP3 Adjust the FL4PMT Voltage until the mean of the peakis at the channel recommended on the APC Beads Target Card STEP 4 Select Time Delay Calibration under the Cytometer menu Click Calibrate
16. This bypasses the sheath filter and allows fluid to travel from the sheath reservoir directly to the flow cell then to the waste reservoir Remember to flip the switch up to pressurize the reservoir FACSCalibur System User s Guide VENT VALVE R R PRESS TO RELIEVE PRESSURE Saline Filter port sheath tubing Figure 7 2 Bypassing sheath filter A CAUTION Bleach run through the sheath filter will break down the filter paper within the filter body causing particles to escape into the sheath fluid and possibly clogging the flow cell 5 Press the HI sample flow rate button and install a tube containing 3 mL of the 1 10 bleach solution on the SIP Press the RUN fluid control button and allow the solution to run for 20 to 30 minutes La Chapter 7 Cleaning and Maintenance 6 Remove the tube of bleach from the SIP 7 Repeat steps 3 through 6 using distilled water instead of the 1 10 dilution of bleach 8 Replace the original sheath reservoir 9 Reconnect the sheath filter by pushing the connector into its fitting until you hear a click l 0 PRIME the flow cell two times Press the PRIME fluid control button to force the fluid out of the flow cell and into the waste reservoir Once drained the flow cell automatically fills with sheath fluid After completion the STNDBY button turns on Repeat this process l Run a tube of distilled water for 5
17. an accessory device you can use with the FACSCalibur flow cytometer to collect and concentrate sorted cells This option is a complete unit with a removable concentrator vessel and waste reservoir Cell Concentrator Module Components When the Cell Concentrator Module option Figure 6 1 is attached to the FACSCalibur cytometer collection station you can sort directly into a cell culture insert or onto a filter within the concentrator vessel The module is equipped with a waste reservoir to collect the excess sheath fluid that has been removed from the sorted cell suspension The sort line for the Cell Concentrator Module is attached to the FACSCalibur cytometer at the third collection port located on the far right You can sort into the module or into the two remaining collection ports waste tubing concentrator air supply tubing CED ss Figure 6 1 Cell Concentrator Module option 105 Chapter 6 Cell Concentrator Module Option 106 air pressure adjustment knob Control Panel The Control panel is installed in the cytometer collection station As Figure 6 2 illustrates the power button air pressure button and air pressure adjustment knob for the Cell Concentrator Module are located on this panel power button air pressure button Figure 6 2 Control panel Power button This button turns on the power to the Cell Concentrator Module When the power is on the cytometer autom
18. at a higher value on the axis channel number B00 1000 Tm a p i T man Figure 3 9 Adjusted FSC and SSC Notice Lin is selected in the Mode pop up menu for side scatter This allows an adjustment of the amplifier gain anywhere between 1 00 and 9 99 Detector voltages are used to make coarse adjustments while amplifier gains are used to fine tune settings Adjust amplification by clicking the up and down arrows or by clicking the icon between the arrows to display a slider FACSCalibur System User s Guide OPTIONAL EXERCISE To further understand how adjusting voltages and amplifiers affects data display do the following Change forward scatter to E01 Notice how the dots move to the right of the display You have amplified your signal tenfold The light signals from the cells can be multiplied by the settings below e E00 multiplies the signal by 10 or 1 e E01 multiplies the signal by 10 or 10 e E02 multiplies the signal by 107 or 100 e E03 multiplies the signal by 10 or 1000 e E 1 multiplies the signal by 107 or 0 1 E01 E02 and E03 are useful for increasing the signal of small events E 1 is useful for reducing the signal of large events Make sure you return the settings to E00 before you proceed gt The next step is to adjust FL1 FL2 and FL3 detectors 17 Repeat steps 4 5 and 6 to create an FL1 vs FL2 dot plot and an FL2 vs FL3 dot plot in the Experiment windo
19. electronic signals These electronic signals are then converted to digital values that are sent to the computer FSC optical signals are detected and converted to proportional electronic signals by a photodiode SSC and fluorescent optical signals are detected and converted to proportional electronic signals by PMTs Manipulation of the signals such as increasing or decreasing them is done by adjusting the pre amplifier level for FSC and the PMT detector voltages for SSC and fluorescent signals Signals are then 2 5 keyboard FACSCalibur System User s Guide processed through linear or logarithmic amplifiers Linear amplification allows signals to be amplified 1 00 to 9 99 times and is useful for applications where analysis of a small range of signal is required ie DNA analysis The 4 log fixed amplifier is used to analyze signals with a wide range of intensity such as those found in immunophenotyping applications FACStation Data Management System The FACStation system Figure 2 6 uses a Macintosh computer that is installed by your BDIS Field Service Engineer Refer to the Getting Started manual that came with your system for additional information on how to set up the Macintosh Complete the Macintosh Basics tutorial that is on the hard drive if you are new to using the Macintosh For more detailed information on using the Macintosh refer to the appropriate Macintosh user s guide printer computer Figure 2 6 FACS
20. from the cytometer Disconnect the air supply tubing from the cytometer Squeeze the metal clip on the quick disconnect and pull the connector from the fitting Disconnect the waste tubing from the waste reservoir Squeeze the metal clip on the quick disconnect and pull the connector from the fitting Remove the sort line from the top of the vessel by pulling the tubing off the needle Slide the small rubber protection cap over the sort line needle at the top of the concentrator vessel FACSCalibur System User s Guide Open the vessel remove the cell culture insert filter holder and pour 50 mL of the cleaning solution into the bottom of the vessel Use either 70 ethanol or 10 bleach Close the vessel and shake vigorously for 1 minute Figure 6 7 Cleaning concentrator vessel Reconnect the waste tubing to the concentrator waste reservoir Push the connector until you hear it click 129 Chapter 6 Cell Concentrator Module Option 130 9 10 11 12 13 Lift the concentrator vessel above the waste reservoir Open the vessel just enough to allow the solution to drain from the vessel Alternately you can reconnect the sort line and the air supply tubing and turn on the Concentrator Module and pressure This pressurizes the vessel and forces the solution into the waste reservoir If bleach is used as the cleaning solution repeat step 6 through step 9 twice using distilled water to rinse th
21. minutes before running patient samples 138 To FACSCalibur System User s Guide Periodic Maintenance There are several instrument components that should be checked occasionally and cleaned as necessary The frequency will depend on how often the instrument is run Other components should be checked periodically for wear and replaced if necessary Changing the Sheath Filter The sheath filter Figure 7 3 located between the sheath reservoir and waste reservoir filters the sheath fluid as it comes from the sheath reservoir Increased debris appearing in an FSC vs SSC plot may be an indication that the sheath filter should be replaced BDIS recommends changing the sheath filter every 3 to 6 months l Open the fluidics drawer Push the vent toggle switch in the direction of the arrow This releases the pressure from the sheath reservoir 139 Chapter 7 Cleaning and Maintenance i Output quick disconnect vent port We O rin 9 filter D aie lt gt base Cc a input quick disconnect Figure 7 3 Sheath filter 3 Squeeze the metal clips of the output and input quick disconnects This releases each quick disconnect Disconnect the air vent tubing from the filter by unscrewing the fitting from the filter vent port The filter should be free of the instrument and the air vent tubing should remain attached to the instrument 140 FACSCalibur System User s Guide 5 Unscrew the base of the filter to rem
22. nieG yh wl oe GALE Se Le ee ea aa sed Re ede doten 58 4 Dual Threshold sated utows slew eee bes sek E ek eo ee Cas esa cee 59 4 4 Setting up the FACSCalibur Instrument for Four Color Analysis 59 Tarmac on the Red Diode Lanie deki aa ena Las hs See a 60 Sectine Wp tne Fy Parmicier 4 osc etietscu eta iio eon inden eovaaseaakes 69 iz Chapter gt Sorong Optone cos bo nese begins Ree e e a bees 75 soning withthe RAC SC alibur System Junkes seams cients a eee hee eae enne ss coed 7 Choosing a Sor Mode 4 agarhtadschd bare bene devas seeder e Sees bane cedeae ot 78 Sal Prune the sent Winey on404 406 id oNeh so eee een ed hanes eS eee aoa ieee 82 J2 Prepare Collection Tubes er nned neds cere cud cee swe ae ARS eee 85 To CEE SO a gases aot aE he gh ee eel eb ay ae 86 IA sSelechie 2 Sort Gale i 25 6 bec ueies used cae beo eda chau ecw dead uae ra stews 88 sine the Sore Counters W Indone 244 2403 bus oe acai ee oe ew aaa ee 90 D SOTUNS Te Sample ack e 4 and Se en Se ee eRe Roe Pekar eee les 91 7 RINGING SOMING sy ranae r By Saxe Bee eee E a E 92 o Reovee Sorted Cells go aire nails uo aid viet Gh oS ht Anaad a nis oe aie Balin dean 4 93 5 Cleaning tie Sor Bing kaare e ehasnei en toead eae sasha 94 DLO GASEPUC SOTUNG 6 iat cate aded whe ctvied oti aes pene sme oamete hes Sie 98 Chapter 6 Cell Concentrator Module Option 2200008 103 OT CelhConcenwator Module Components entere rn a E secs ate te y
23. reservoir with distilled water then fill with sheath fluid from another or new lot bulk container SORTING Sorted sample not flowing into collection tubes or no fluid flows into collection tubes after pressing sort line purge button Cause Solution Cause Solution Cause Solution Cause Solution Sort line clogged Perform the sort line cleaning procedure outlined in Section 7 2 2 Sort gate not set Set sort gate Section 5 6 or select gate from Sort Gate field in Sort Setup window Sort events too high Dilute sample RUN fluid control button not activated Press the RUN fluid control button 157 Chapter 8 Troubleshooting SYMPTOM SYMPTOM SYMPTOM 158 Cells not viable after sorting Cause FACSFlow used as sheath fluid Solution Use phosphate buffered saline calcium and magnesium free for the sheath fluid when sorting cells Cause Collection tubes not coated with 4 BSA Solution Coat collection tubes with BSA leave small amount in bottom of tube to cushion cells Cause Cells not viable before they were sorted Solution Check viability before sorting Cell recovery low Cause Collection tubes not coated with 4 BSA Solution Coat collection tubes with BSA Cause Centrifugation technique Solution See Section 5 8 for information on centrifugation Depending on the cell type and concentration this recovery procedure may need to be adjusted Low purity
24. target cells FACSCalibur System User s Guide The sort envelope is the area within the sample stream that the catcher tube collects as it captures a target cell The size of the envelope reflects the amount of time the catcher tube remains in the sample stream to capture the cell When this envelope contains the target cell it can also contain a nontarget cell This results in a conflict should the catcher tube sort a cell if a nontarget cell will be sorted along with it The sort mode determines whether or not to sort a cell when a conflict occurs Figure 5 2 illustrates how the system decides to sort a cell for each sort mode Use the Sort Setup window described in Section 5 4 to select the appropriate sort mode for a particular sorting application je jam mo eje e Rm Oo oe ge ge no sort O oO sor gt sort no sort Single Cell Recovery Exclusion Figure 5 2 How envelopes are sorted for each sort mode 79 Chapter 5 Sorting Option 80 Single Cell In Single Cell mode a sort occurs whenever a single target cell is identified in the envelope If an additional cell even a target cell is located within the sort envelope the envelope will not be sorted The result is high purity with less emphasis on recovery Single Cell mode also gives increased count accuracy Recovery In enhanced Recovery mode a sort occurs whenever an envelope is identified as having a target cell even i
25. threshold 59 using FSC 59 Lf Index 178 e electrical safety x electromagnetic compatibility x7 electronics system 24 Exclusion 80 Experiment document 28 Export Stats file 27 f FACS Loader 7 FACSCalibur instrument cleaning 131 133 components 4 installation 5 intended use 4 leaving 22 options and upgrades 7 overview 11 preventive maintenance 133 software for use with 7 8 sorting with 77 FACSComp_ 5 26 51 FACSConvert 5 27 FACS Loader 7 FACStation Data Management System 4 2527 converting files 5 27 Experiment documents 28 filing system 27 hardware 4 reports 28 software 5 filter membrane 107 108 insert 108f preparing 110 files export stats 27 list mode data 27 filter air 143f filters long pass 24 short pass 24 FITC see fluorescein 33 49 FL4 optics 55 56f FL4 option 7 55 dual threshold 59 instrument setup 59 optics 55 56f FL4 parameter setup 70 74 flow rate buttons 12 PRIME 138 RUN 137 142 STNDBY 138 fluid control panel 12f flow rate buttons 12 fluid detection probe cable 15 fluidics 12 priming 21 22 fluidics drawer 14f 15 fluorescein FITC 33 49 fluorescence overlap 33 forward light scatter FSC 24 43 four color acquisition 55 four color analysis 59 74 FACSCalibur instrument setup 59 63 FSC forward light scatter 24 43 g h i hardware 4 in vitro diagnostic 4 installation 5 instrument controls accessing 31 adjusting 31 instrument settings 61 ampli
26. 11 10823 02 Rev A FACSCalibur System User s Guide 02 61760 02 August 1996 DICKINSON Becton Dickinson Becton Dickinson Becton Dickinson Nippon Becton Dickinson Becton Dickinson Immunocytometry Systems Canada Inc European HQ Company Ltd Worldwide Inc 2350 Qume Drive 2464 South Sheridan Way Denderstraat 24 DS Bldg 30 Tuas Avenue 2 San Jose CA 95131 1807 Mississauga Ontario B 9320 Erembodegem Aalst 5 26 Akasaka 8 chome Singapore 2263 Ordering Information 800 223 8226 L5J 2M8 Belgium Minato ku Tokyo 107 Tel 65 861 0633 Customer Support Center Canada Tel 32 53 720211 Japan FAX 65 860 1590 800 448 2347 BDIS Tel 905 822 4820 FAX 32 53 720450 Tel 81 3 5413 8251 FAX 408 954 2347 BDIS FAX 905 822 2644 FAX 81 3 5413 8155 Copyright Disclaimer Trademarks Limitations FACSCalibur User s Guide Becton Dickinson and Company 1996 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 the prior written permission of Becton Dickinson Immunocytometry Systems BDIS 2350 Qume Drive San Jose CA 95131 United States of America BDIS reserves the right to change its products and services at any time to incorporate the latest technological developments This guide is su
27. 4 in the Detector column Figure 4 4 62 FACSCalibur System User s Guide Param Detector Yoltage Amp Gain Mode Param Detector Yoltage Amp Gain Mode PI FSC oof PI FSC 1 00 f P2 ssc 150 oo f P2 ssc 150 1 00 fj P3 FLI 150 o0 f P3 FLUI 150 1 00 P4 150 oo 3 P4 FL2 150 1 00 fj P5 FL3 150 oo f P5 FL3 150 1 00 3 P6 FL2 00 3 Lin P6 FL2 1 00 Lin P7 FL2 00 3 i ZO O FLZeW saoj ti p7 fis i58 ih P7 FL4 150 CO Four Color DDM Param E Four Color DDM Param Figure 4 4a Four Color off Figure 4 4b Four Color on OPTIONAL EXERCISE Using the DDM Param pop up menu on the Detector Amps window choose FL4 as the DDM parameter on the Detectors Amps window Figure 4 5 Two P7 lines appear on the Detectors Amps window One line will be disabled gray depending on DDM parameter choice Param Detector Yoltage Amp Gain Mode P1 FSC t EF 1 00 ee 00 f P2 ssc 150 1 00 3 sh oo P3 FLI 150 1 00 cid oo f P4 FL2 150 1 00 3 PA 00 f FL3 150 1 00 3 00 Lin FL4 A os 8 ig Lis FL4 W PEG FL1 EJ Four Color I FL2 p pen gt Four Color DDM Param FL4 Figure 4 5a DDM Param pop up menu Figure 4 5b FL4 chosen as DDM parameter This is the method you use to select Pulse Processing of the FL4 parameter 63 Chapter 4 FL4 Option When Four Color is checked in the Detectors Amps window DDM parameter selections are FL1 FL2 FL3 and FL4 The area of the selec
28. E 4 1 2 Components of the Basic FACSCalibur System 4 x 0ieus 24d a3 4 deele tk Shae eee s 4 Dao NO tetra eee ee eee Ree ee Oe ea ee eae 5 LA Ophion and perades secu e e e ote d orescence seccenemau sameness aoe fi Chapter 2 Genine Started ste taney aea ee ealaee tee netda seh abun e ness 9 21 FACSCalibur istrument Overview sci anaes cee ee ae eee ewes Aes Y 11 22 Fluidos Drawer Components gs n2 ae occy ee eeene we ota ee bene eoan ane 14 Pilne tae Sheath Reser Ot ereina i on eds ee O E atvielad dees 16 Emptying the Waste RK esenvOir awed og 28s Gacy Gc ed seein ee eae ee wae os ke 19 Prine the Fligics 12 ou vecuee iets Foes as oO ee Ba awed Rae ere aie Sees 21 Leaving the PAC S alibur Instrume sinen ieee dies ku aan aewopetesdaaneuene 22 22 Optical yem Com ponents ss10cce deg a40 9 beau nee oe eee se eee 23 2A TENGCHROMICS SV StCUN a aa dpa tee a r a a AT 24 2 FACStation Data Management Systemi sses ran roce E A A dada 25 Chapter 3 Instrument Setup for Acquisition of Samples 29 3 1 Accessing nstrument Controls in CELLQuest a n ce eed ee ed eed ae eS 31 32 Opumiizme the Instrument Seins e ost oes waee Swaceenaden ee sh ate ew beak es 35 Oo sayine the Insthuiment Scns nc na cieneodsoea tuecuedt hie auiie ss hue sae ede es 51 Chapters FLAO pion 3 64655 2358s chee Cg cea eee eee 53 AN Opus eerop er pike tdad AEE as dee er ew outa daa teed ehep aoe a 55 Bed Pime Delay ElCCtIONICs seriens 2
29. Quest Software Users Guide for detailed instructions on using the various features of an Experiment document 36 FACSCalibur System User s Guide 3 Choose Connect to Cytometer from the Acquire menu Acquire Acquisition amp Storage Parameter Description Keywords Counters Edit Reagent List Edit Panels Connect to Cytometer 6 The Acquisition Control window appears File I Setup Communication between the computer and cytometer is established and the cytometer menu is active giving you access to the instrument controls The Acquire button is active and the Setup box is checked When the Setup box is checked data is not saved Click and drag the window to a clear area of the screen 4 Choose Dot Plot from the Plots menu The Dot Plot dialog box appears Figure 3 4 Use the dot plot to view data while adjusting instrument settings 37 Chapter 3 Instrument Setup for Acquisition of Samples Dot Plot Plot Source x Parameter pl 256 vj YT Parameter _p2__256 j Gate _No Gate v Show 100 vw of Dots Single C Show 00g i Bets D Multi Color Gating Titl Bsn Figure 3 4 Dot Plot dialog box 5 Choose Acquisition from the Plot Source pop up menu Figure 3 5 Click and hold the Plot Source box in the Dot Plot dialog box to open the pop up menu 6 Choose FSC for the X parameter and SSC for the Y parameter Click and hold each parameter box to open a pop up me
30. TIONS Performance Fluorescence Sensitivity Fluorescence Resolution Forward and Side Scatter Sensitivity Forward and Side Scatter Resolution Excitation Optics Optical Platform Lasers Beam Geometry Emission Optics Optical Coupling Background Rejection Forward Scatter Detector and Filter Side Scatter Detector Fluorescence Detectors and Filters Estimated detection limit is 750 molecules of equivalent soluble fluorescein Coefficient of variation in FL2 Area of lt 3 full peak for propidium iodide stained chicken erythrocyte nuclei Sensitivity enables the separation of fixed platelets from noise Scatter performance is optimized for resolving lymphocytes monocytes and granulocytes Fixed optical assembly 15 milliwatt 488 nm air cooled Argon ion laser life expectancy gt 5 000 hours Optional second laser nominally 635 nm Prismatic expander and achromatic spherical lens provide 22 Um x 66 um elliptical beam for argon ion laser and nominally 15 um x 61 um elliptical beam for red diode laser Quartz cuvette is coupled to emission lens by refractive index matching optical gel for optimum collection efficiency Obscuration blade and slit minimize unwanted laser radiation at the detector High performance solid state silicon detector with 488 nm band pass filter for clear signal detection and red diode laser signal rejection High performance photomultiplier using Brewster angle beam splitter in the em
31. al Environment It shall not be used in the residential commercial and light industrial environment unless the apparatus also conforms to the relevant standard EN 50081 1 xi Safety and Limitations xii CHAPTER 1 Introduction CHAPTER 1 Summary gt introduction intended use components of basic system hardware and software installation options and upgrades FACSCalibur System User s Guide The FACSCalibur system is a modular benchtop flow cytometer from Becton Dickinson Immunocytometry Systems BDIS It consists of a sensor module a computer module and various software packages Designed for applications that range from routine clinical to advanced research this system analyzes cells as they pass one at a time through a focused laser beam The FACSCalibur system can measure several parameters including forward light scatter FSC side light scatter SSC and several fluorescence parameters as well as the pulse area and width of any fluorescence parameter Figure 1 1 FACSCalibur flow cytometry system ll Chapter 1 Introduction 1 1 Intended Use The FACSCalibur flow cytometer is an in vitro diagnostic product for enumerating leucocyte non blast subsets with the appropriate software See the relevant software user s guide or reagent package insert for in vitro diagnostic instructions In addition the FACSCalibur system can be used for many research applications including mult
32. allows for aerosol free sterile sorting DATA MANAGEMENT SYSTEM FACStation Power PC Reduced Instruction Set Computing RISC CPU running at 120 Mhz clockspeed 40 MB RAM 256 kilobytes 1 2 gigabyte hard disk On board Ethernet built in AppleTalk Networking and Apple File Sharing 4x Speed CD 600i Apple Vision 1710 17 inch Trinitron tube Flow Cytometry Standard FCS 2 0 ASCII results file for data export 73 Appendix B FACSCalibur Specifications Power Water Supply Air Supply Temperature Humidity Air Filtering Lighting Size Weight REMOTE DIAGNOSTICS Remote diagnostics modem provided for direct customer support instrument interaction INSTALLATION REQUIREMENTS US 120 VAC 10 50 60 Hz 2 Hz Current 20 amps maximum Outside US External transformer needed for 100 VAC 10 50 60 Hz and 220 240 VAC 10 50 60 Hz 2 Hz None required None required OPERATING ENVIRONMENT 16 29C 60 85F 10 90 relative non condensing Excessive dust and smoke must be avoided Optics and detectors are shielded from room lighting Sensor module width 91 4 cm 36 in depth 61 5 cm 24 2 in height 67 3 cm 26 5 in 124 5 49 in with cover open Computer 48 x 41 x 54 cm 19 L x 16D x 23 H Printer 48 x 41 x 54 cm 19 L x 16 D x 23 H Sensor Module 109 1 kg 240 lbs Computer 50 kg 110 lbs Specifications subject to change without notice 174 Index 176 a acquire bu
33. atically sorts into the concentrator vessel instead of the collection tubes When the module is turned on the air pressure LCD displays 000 Air pressure button This button turns on the air pressure to the concentrator vessel You must turn on the power to the Cell Concentrator Module before you can turn on the air pressure When you turn on the air pressure the LCD displays a number somewhere between 000 and 999 FACSCalibur System User s Guide Air pressure adjustment knob Use this knob to adjust the air pressure within the concentrator vessel When you increase the air pressure in the vessel you increase the rate at which fluid flows through the filter membrane or cell culture insert The LCD displays the voltage applied to the valve that regulates the air flow Concentrator Vessel This removable vessel is designed with upper and lower compartments separated by the cell culture insert holder or filter holder Sorted cells are deposited into the cell culture insert or filter holder through the sort line that extends to the insert Filtered air is transported through tubing into the upper compartment to pressurize it The lower compartment collects the sheath fluid as it passes through the membrane A waste tube carries fluid to the waste reservoir Figure 6 3 illustrates how this process occurs A magnet located at the bottom of the vessel ensures the vessel remains securely in place within the Cell Concentrator Module base or wh
34. ause Solution Contaminated sample Prep specimen again making sure tube is clean Scatter parameters appear distorted Cause Solution Cause Solution Cause Solution Cause Solution Cause Solution Cause Solution Instrument settings adjustment is necessary Perform optimization procedure Air bubble in flow cell Press PRIME to drain and fill the flow cell See Section 2 2 3 step 2 Air in sheath filter Vent air from sheath filter See Section 2 2 3 step 1 Flow cell dirty Perform monthly cleaning procedure See Section 7 1 2 Air leak at sheath reservoir Check Status sample voltage with test tube off sample voltage should be 10 2 If less replace sheath tank then replace cap then replace gasket Check connectors on sheath sensor and saline filter Hypertonic buffers Check pH of buffers and fixative Excessive amount of debris appearing in display Cause Solution Threshold level too low Increase threshold level SYMPTOM Cause Solution Cause Solution Cause Solution Cause Solution FACSCalibur System User s Guide Sheath filter dirty Change sheath filter See Section 7 2 3 Flow cell dirty Perform monthly maintenance see Section 7 1 2 or wash with 10 bleach and warm deionized water Sample contains excessive amount of debris Examine sample under a microscope Stock sheath fluid contaminated Rinse sheath
35. ay not be necessary to coat filter membranes with BSA filter forceps FACSCalibur System User s Guide 2 Place the white O ring into the top of the holder 3 Secure the top of the filter onto the bottom piece Avoid crimping the filter If it is not seated in the holder remove the top and adjust the filter membrane fi Figure 6 5 Securing top of filter holder III Chapter 6 Cell Concentrator Module Option 6 3 Sorting with the Cell Concentrator Module The following procedure shows you how to sort using the Cell Concentrator Module option using a cell culture insert to collect the sorted cells Priming the Sort Line The sort line must be clear before you begin a sort To check that the sort line is clear do the following l Assemble the concentrator vessel Screw on the top of the vessel by turning clockwise Make sure the entire vessel is sitting securely on top of the Cell Concentrator Module base You do not need to have any filter inserts installed in the vessel at this time Press the power button The LCD displays a value of 000 Make sure that there are no tubes in the first two collection ports starting from the left before pressing the sort line purge button 112 FACSCalibur System User s Guide 3 Install a tube of PBS on the SIP and set the fluid control to RUN A Press the sort line purge button See Section 5 1 Priming the Sort Line If there is no clog in the sort line you sh
36. bject to change without notice BDIS welcomes customer input on corrections and suggestions for improvement Although this guide has been prepared with every precaution to ensure accuracy BDIS assumes no liability for any error or omission nor for any damages resulting from the application or use of this information FACS and Falcon are registered trademarks of Becton Dickinson and Company FACSCalibur CELLQuest FACSComp FACSConvert CONSORT FACSFlow CaliBRITE SimulSET Attractors PAINT A GATE O FACStation and FACSNet are trademarks of Becton Dickinson and Company Macintosh Apple and the Apple logo are registered trademarks of Apple Computer Inc ModFit L7 is a trademark of Verity Software House Inc Please refer to the appropriate reagent package inserts and software user s guides for specific instructions and limitations on in vitro diagnostic use The Sorting option the FL4 option and the Cell Concentrator Module option are for research use only Use of controls or adjustments or performance of procedures other than those specified in this user s guide may result in hazardous laser light exposure FACSCalibur System User s Guide Table of Contents PrE IEE soe os Selec Fue ah oe ta ee peo ees res E are Green we A EE ait aires ene ne ete v Sakean EMAON ra e Seven aN a a a aaa a A a a EA ix Chapter 1 Introduccion srete sie e peoe aE Rn E a es 1 eA tended Uses ance nea rs os kee aR eeher eases ereei care E
37. chcock to the closed position 2 Remove the tube of distilled water from the SIP 3 Clear the flow cell of trapped air bubbles by priming it Press the PRIME fluid control button to force the fluid out of the flow cell and into the waste reservoir Once drained the flow cell automatically fills with sheath fluid at a controlled rate to prevent bubble formation or entrapment The STNDBY button is orange after completion 4 Replace the distilled water tube on the SIP Place the support arm under the tube Leaving the FACSCalibur Instrument When you walk away from the system press the STNDBY fluid control button to stop sheath consumption and reduce laser power Install a tube containing no more than 1 mL of distilled water on the SIP and center the tube support arm This prevents the sample injection tube from drying out FACSCalibur System User s Guide A CAUTION Some fluid backflows in STNDBY mode be sure the tube left on the SIP contains no more than 1 mL of distilled water This will prevent fluid from overflowing into the air supply tubing that pressurizes the tube 2 3 Optical System Components Figure 2 5 is a simplified diagram of the optical system used in the FACSCalibur FL1 488 10 FL 985 42 90 10 beam splitter DM 5605P FL3 Wie fluorescence collection lens blue laser A88 nm a Fee d 488 10 SC diode focusing lens Figure 2 5 FACSCalibur optical system 23 Chapter 2 Getting Starte
38. clockwise 123 Chapter 6 Cell Concentrator Module Option Disconnect the upper tubing of the sheath filter from the SALINE FILTER port Squeeze the metal clip on the quick disconnect and pull the connector from the fitting Connect the tubing from the syringe to the port labeled SALINE FILTER Push firmly until you hear a click VENT VALVE fR R PRESS TO RELIEVE PRESSURE Saline Filter port 7 Install a 50 mL tube in the first collection port 124 FACSCalibur System User s Guide 8 Place a tube of distilled water on the SIP 9 Set the fluid control to RUN l 0 Press the sort line purge button l l Slowly yet firmly apply pressure to the syringe plunger Depress the plunger until approximately 10 mL of fluid has been dispensed from the syringe and has entered the collection tube Once the button is pressed the valve remains open for approximately 30 seconds Press the button again if you have not dispensed all 10 mL of fluid l 2 Remove the collection tube from the first collection port far left position and place it in the second collection port 1 3 Repeat steps 4 and 5 123 Chapter 6 Cell Concentrator Module Option l 4 Remove the collection tube l 5 Turn on the Cell Concentrator Module by pressing the power button l 6 Rep
39. ction 5 1 Priming the Sort Line for more details l l Apply pressure slowly yet firmly to the syringe plunger Depress the plunger until approximately 10 cc of fluid has been dispensed from the syringe and has entered the collection tube l 2 Remove the collection tube from the leftmost position and place the same tube in the middle position 13 14 15 16 17 FACSCalibur System User s Guide Repeat steps 10 and 11 for the middle position Remove the collection tube from the middle position and place the same tube in the position on the far right Repeat steps 10 and 11 for the position on the far right Remove the syringe from the upper sheath fluid connector and reconnect the sheath fluid tubing Prime the flow cell 2 to 3 times to remove any air that may have entered the flow cell See Section 5 1 for information on priming _ NOTE Ifa clog is still apparent after you complete this procedure perform the procedure again using a 1 10 dilution of bleach in the syringe followed by distilled water 97 Chapter 5 Sorting Option 98 5 10 Aseptic Sorting The FACSCalibur system can sort cells to be used for culture or functional studies To meet the needs of this application sorting requires a clean environment to keep the sorted sample free from contaminants when put into culture Perform all steps of your preparation procedure using an aseptic technique l Prepare the following sterile solutions
40. ction tubes on the instrument Starting at the first collection port place from one to three BSA coated 50 mL conical collection tubes into the collection station The instrument 8 amp 5 Chapter 5 Sorting Option detects the number of tubes installed and fills each tube starting with the one on the left It takes 9 minutes to fill each tube with 40 to 45 mL of fluid first collection port 5 3 Creating a Sort Gate Gates defined in CELLQuest software can be used for acquisition analysis and sorting For detailed information on drawing a region or creating gates refer to the CELL Quest Software Users Guide l Create an acquisition plot FACSCalibur System User s Guide 2 Choose Connect to Cytometer from the Acquire menu The Acquisition Control window appears The Setup box should be checked File K Setup Restart J Save J _Abort_ Install the sample tube on the SIP quickly center the tube support arm under the tube and press the RUN fluid control button 4 Click Acquire in the Acquisition Control window View the appropriate plots to ensure the instrument settings have been properly optimized See Section 3 2 Optimizing Instrument Settings for more information Make adjustments if necessary 5 Click to select a region tool in the tool palette Choose among rectangular elliptical polygonal or histogram regions 87 Chapter 5 Sorting Option Click in the plot and dra
41. d 2 4 The argon ion laser in the FACSCalibur instrument produces 15 mW of 488 nm light This beam provides a spot that is large enough for most cells to be entirely illuminated within the beam when they intercept the beam and also large enough to give relatively uniform excitation across the sample stream As the focused laser beam interacts with a cell with fluorescent markers scattered light and fluorescence signals are created at the same time The forward scatter FSC signal is collected by the forward scatter diode The side scatter SSC and fluorescence parameters are collected by the 90 degree collection lens and focused into a series of optical filters The collected light is spectrally split by a collection of dichroic mirrors DM and filters The first mirror 560 SP Short Pass encountered passes green and yellow green fluorescence and reflects longer wavelengths The passed light goes to the FL1 green yellow green photomultiplier tube PMT with a 10 fraction split off to provide the side scatter signal to the next PMT The reflected light goes back to a second mirror 640 LP Long Pass that passes long wavelength red light to the FL3 PMT and reflects the yellow and orange light to the FL2 PMT See Appendix B FACSCalibur Specifications for the exact wavelength characteristics of the dichroic mirrors and filters Electronics System The electronics system in the FACSCalibur flow cytometer converts optical signals into
42. de Cc gt O ring L C gt cell culture insert holder See O ring Figure 6 6 Installing cell culture insert holder Place a BSA coated cell culture insert into the insert holder and close the concentrator vessel See Section 6 2 for information on coating the cell culture insert Screw on the top of the vessel by turning clockwise Avoid overtightening Make sure the sort line and waste line are not kinked cell culture insert insert holder 115 Chapter 6 Cell Concentrator Module Option 116 If not already on turn on the Cell Concentrator Module by pressing the power button Be sure the sort line air supply tubing and waste tubing are properly connected The LCD displays a value of 000 Once the Cell Concentrator Module is turned on sorted cells automatically bypass the collection tubes and are collected in the concentrator vessel Install a tube of PBS on the SIP and set the fluid control to RUN Make sure the Setup box in the Acquisition Control window is checked and the Sort Gate in the Sort Setup dialog box is selected before you proceed to the next step Click Acquire in the Acquisition Control window When the insert is filled half way with fluid press the air pressure button to pressurize the concentrator vessel Figure 6 B The LCD displays a value between 000 and 999 i 11 FACSCalibur System User s Guide Adjust the pressure by turning the air pressure adjustment knob F
43. e and any additional software programs you may have purchased will be loaded on your FACStation computer before shipment NOTE For installations outside the US a power transformer conditioner is necessary to accommodate 100 V 10 220 V 10 or 240 V 10 50 to 60 Hz 2 Hz 20 A 3 Chapter 1 Introduction When CELLQuest software is installed before shipment the supporting files are placed in the appropriate folders of the computer AcglibPPl BODPACDriver BOP AC BOF AC Init Performing acquisition using the Macintosh PowerPC requires the presence of the Acquisition Library AcqLibPPC and the BDPACDriver in the Extensions folder BDPAC must be present in the Control Panels folder and the BDPAC Init needs to be in the Startup Items folder Your Field Service Representative will access the BDPAC window during instrument installation to configure CELLQuest software for your cytometer type and to enter the serial number Change the configuration information only if the computer is connected to a different cytometer or if the software is reloaded Refer to the CELL Quest Software Users Guide for help on reconfiguring the BDPAC window _ NOTE CELLQuest acquisition on the Quadra 650 requires only the presence of BDMAC in the Control Panels folder BORA FACSCalibur System User s Guide 1 4 Options and Upgrades FACSCalibur Instrument The basic FACSCalibur flow cytometer comes equipped with up to three color
44. e transfer program such as FACSNet Macintosh or CONSORT File Exchange to transfer HP files to the Macintosh computer See Section 1 3 for optional software available for the FACStation FACStation Filing System If you are new to the Macintosh refer to the Macintosh Users Guide for detailed help in understanding how the Macintosh works Using the installed software with the FACSCalibur flow cytometer you will create documents and files save them in folders and store these folders in designated locations for retrieval at a later time The types of documents and files you create include List mode data files unprocessed data files containing all of the measured parameters for each particle in a sample as well as information describing the sample FACStation software creates and reads list mode files in FCS 2 0 format NOTE FCS 1 0 files can be converted to FCS 2 0 using FACSConvert software Export Stats files TEXT files numbers and letters used to transfer data obtained from an analysis into other applications such as spreadsheet and database programs a Chapter 2 Getting Started 28 Reports PICT files graphics or pictures or TEXT files that contain the results of single tests or groups of tests Instrument settings files files that contain the information necessary to set up the FACSCalibur flow cytometer for a particular application once saved these settings can be retrieved and sent to the cytometer
45. e 14 Bal seal 144 tube support arm 13 14 22 Sample O ring changing 147 148 secondary threshold 33 with FL4 option 33 sensor unit 4 11f fluid control panel 11 fluidics drawer 11 sample injection port SIP 11 13f 134f Setup box 37 43 sheath filter 15 137 139 140f bypassing 137f changing 139 142 pinchcock 15 sheath fluid 107 108 sheath fluid detection probe cable 16 sheath reservoir 15 filling 16 18 sheath tubing 15 16 side light scatter SSC 24 44 SimulSET 8 Single Cell sort mode 80 SIP sample injection port 11 13f 134f software Attractors 8 CELLQuest 5 6 26 31 FACSComp_ 5 26 51 FACSConvert 5 27 Modfit LT 5 PAINT A GATE 8 sort preparing to 82 sort counters 90 Sort Counters window 90 sorted cells recovering 93 94 sort envelope 79 sort gate 86 89 creating 86 88 selecting 88 90 sorting 118 120 aseptic 98 102 concentrating cells 119 ending 92 93 option 7 77 preparation 82 recovering sorted cells 93 94 120 122 sample 91 92 with Cell Concentrator Module 112 117 sorting and concentrating cells 118 119 sort line cleaning 94 97 122 clog 97 113 priming 82 purge button 83 recovering sorted cells from 120 122 sort mode choosing 78 81 Exclusion 80 Recovery 80 Single Cell 80 Sort Setup window 88 sort yield 81 sorted cells recovering 93 94 120 122 removing for re analysis 122 Sorting option 7 77 spectral overlap 33 34f 57f SSC see side light scatter 44 STNDBY fluid control bu
46. e Figure 2 1 is located on the lower left panel of the instrument it slides out for easy access to the fluid reservoirs and sheath filter Before turning on the instrument check the fluid levels of both the sheath reservoir and the waste reservoir The sheath reservoir should be no more than 3 4 full sufficient for approximately 3 hours of run time and the waste reservoir should contain approximately 400 mL of undiluted household bleach which contains 5 sodium hypochlorite The fluidics drawer contains the following Metal bracket prevents sheath tank from expanding while under pressure Ball valve allows tank to pressurize only when metal bracket is in place Air supply tubing supplies pressurized air to sheath tank Sheath tubing carries sheath fluid out of sheath tank Sheath filter removes particles larger than 0 2 microns from sheath fluid Sheath filter air vent tubing vents trapped air from sheath filter Sheath filter pinchcock closes sheath filter air vent tubing Sheath reservoir a 4 L container located on the left and secured by a metal bracket holds enough sheath fluid for approximately 3 hours of run time equipped with a fluid level detector that indicates via the software a near empty condition Waste reservoir a 4 L container located on the right that collects the fluid waste after it flows from the flow cell equipped with a fluid level detector that indicates via the software a near full condition
47. e vessel Wipe the vessel dry and store it on the Cell Concentrator Module base Clean salt deposits and dirt from the vessel screw threads by wiping them with a damp cloth Apply a thin coat of silicon grease to the threads if you have had difficulty opening the vessel Flush the O rings insert holder with the cleaning solution and dry them If bleach is used rinse the O rings and insert holder with distilled water before drying them CHAPTER 7 Cleaning and Maintenance CHAPTER 7 Summary 132 daily FACSCalibur instrument cleaning monthly cleaning changing the sheath filter cleaning the air filter changing the Bal seal changing the sample O ring 7 1 FACSCalibur System User s Guide The FACSCalibur instrument is designed to require minimum maintenance However to preserve the reliability of the instrument you must regularly perform basic preventive maintenance procedures This chapter explains daily monthly and periodic cleaning procedures you should follow to keep your instrument in top performing condition WARNING Blood samples may contain infectious agents hazardous to your health Follow appropriate biosafety procedures wear gloves whenever cleaning the instrument or replacing parts Daily Cleaning NOTE A 5 solution of sodium hypochlorite can be substituted for undiluted bleach in the following cleaning procedures Higher concentrations and other cleaning agents may damage the instrument Whe
48. eat steps 4 and 5 twice You are now rinsing the sort line going to the Cell Concentrator Module This sort line is rinsed twice because the sort line going to the Cell Concentrator Module is approximately twice the length of the sort line going to the first two collection stations 1 7 Remove the syringe and reconnect the sheath filter tubing l g Press the air pressure button to turn it on The LCD displays a value between 000 and 999 The pressure will force the liquid out of the bottom chamber of the concentrator vessel and into the waste reservoir 126 FACSCalibur System User s Guide l 9 Press the air pressure button to turn if off 2 0 Press the power button to turn off the Cell Concentrator Module 2 l Drain and fill PRIME the flow cell two to three times to remove any air that may have entered the flow cell 2 2 Leave the instrument in STNDBY mode with a tube on the SIP containing no more than 1 mL of distilled water Cleaning the Concentrator Vessel The concentrator vessel should be cleaned and decontaminated after use and before being stored at the end of the day using either 70 ethanol or 10 bleach solution as cleaning solutions Undiluted bleach contains 5 sodium hypochlorite A CAUTION The concentrator vessel and insert holders should not be autoclaved boiled or exposed to high temperatures J27 Chapter 6 Cell Concentrator Module Option 128 l 2 5 Disconnect the concentrator vessel
49. ector of FL2 fluorescence overlap Notice the PE labeled beads move toward the x axis FL3 Continue to adjust until the entire population is below the horizontal marker line Figure 3 15 49 Chapter 3 Instrument Setup for Acquisition of Samples Beads O07 Figure 3 14 Adjusted FL1 FL2 compensation Beads 005 Beads 008 Figure 3 15a Unadjusted compensation Figure 3 15b Adjusted compensa 27 Check compensation for the PerCP bead population Since PerCP fluoresces far in the red range there is usually no PerCP fluorescence overlap into the FL2 or FL1 detectors thus there is generally no need to adjust compensation This may not be true for other fluorochromes 50 FACSCalibur System User s Guide You have now completed the instrument adjustments necessary for you to view and analyze data This procedure is similar to what FACSComp does automatically When you acquire biological samples BDIS recommends you optimize instrument settings with these samples after you run FACSComp 3 3 Saving the Instrument Settings Instrument settings can be saved so you can retrieve them to practice adjusting them or you can retrieve them for use at another time Ji Choose Instrument Settings from the Cytometer menu The Instrument Settings window appears Instrument Settings Cytometer Type FACSCalibur Detectors Asps Param Detector Voltage AspGain FSC E00 1 00 SSC 150 FL1 150 150 Displaying Current Status
50. ee 105 6 2 Preparing the Cell Concentrator Module to Sort cc24 ces gn eeieemast eee esena 108 6 3 Sorting with the Cell Concentrator Module 214 sos asedecions eed eaie isa ees 112 Prine tie Sore E Csin a ale are ee ele Ea a ee hens 112 Determine Reference Pressures cesi eein a r A hewn dees 113 Sorting and Concentrating Cells mrrpisrsearad a eea ae OA 117 Recovering Sorted Cells from the Sort Line 2 0 ee ee 120 Removing Cells tor Re anal ysis lt tc e het eota ued yous cen eke Patetneen 121 Cleaning the Son Pine iota ead a arnte ened sacha Bees pe meee Reads 122 Cleaning the Concentrator Vessel eiks oun eeeeneeren teams ena deeb ok bones 127 Chapter 7 Cleaning and Maintenance 0 cee eee ee eee eens 131 TN Dail Gleaming osx va oe aude c cae ieee eee eRe eet ates 133 i Momi Cleaning gaera aaa opr aswoe ies E E E a A ele oon pies 135 Feo Penodi Niaintenance s5 32 4 ces h ace wand 1a G Le arte hed ee ba ed balee dae 139 Changing the Sheath Pie erine cia na esses Get igang oak 139 Cean Dene Pate Fit ter aa 9 Ses a 25 she A E Sa cath eters Ges ae a a 143 Chaneime the Bal Scdlic Avance ny ecu EREA rate EA EE OI EENT e 144 Chaneine the sample OTN cae toda 4 ade Gee ad ee setae eda doe decease 147 FACSCalibur System User s Guide Chapter 8 Troubleshooting oi 4 5 42 0 p 3 0 be eases baa mete beh asses bee ays 149 Appendix A Consumables and Service Information 4 163 Appendix B FACSCalibur Specifications
51. ells to travel from the laser intercept to the catcher tube is constant When the decision is made to capture the target cell the electronics waits a fixed period of time to allow the cell to reach the catcher tube and then triggers the catcher tube to swing into the sample stream to capture the cell Figure 5 1a shows the catcher tube in its resting position in the sheath stream Figure 5 1b shows the catcher tube positioned in the sample core stream ready to capture a target shaded cell Za Chapter 5 Sorting Option 78 catcher tube Figure 5 1a Catcher tube in sheath stream Figure 5 1b Catcher tube in sample stream Because the catcher tube is positioned in the sheath stream while it waits for a target cell it continuously collects sheath fluid along with the sorted cells This results in a dilute sorted sample For further processing or reanalysis after sorting concentrate the cells by using a centrifuge See Section 5 8 Recovering Sorted Cells for instructions The Cell Concentrator Module option concentrates cells as they are being sorted See Chapter 6 of this user s guide for instructions on using this option Choosing a Sort Mode Choose a sort mode based on the composition and concentration of the sample suspension as well as on the objectives you wish to achieve with the collected cells When sorting a rare population for example you may have to sacrifice purity in order to sort the maximum possible number of
52. en placed in the cytometer collection station sorted sample is introduced through the sort line and deposited l Air is pumped into the upper chamber of the vessel to pressurize it into cell culture insert Excess sheath is forced into the lower chamber and carried off to the waste reservoir Figure 6 3 Concentrator vessel 107 Chapter 6 Cell Concentrator Module Option Waste Reservoir This 1 liter reservoir see Figure 6 1 collects the fluid that is removed from the sorted cell suspension This excess sheath fluid which passes through the insert membrane is deposited into the lower compartment of the concentrator vessel before being carried to the waste reservoir A 0 22 um filter allows aerosol free air to escape from the reservoir Empty the waste reservoir whenever you fill the cytometer sheath reservoir 6 2 Preparing the Cell Concentrator Module to Sort As Figure 6 4 illustrates there are two ways to recover cells from the Cell Concentrator Module The first is to insert a filter membrane onto a filter holder the second way is to use a cell culture insert filter membrane cell culture insert insert holder filter holder Figure 6 4a Cell culture insert Figure 6 4b Filter membrane insert 108 FACSCalibur System User s Guide Before you begin your sort it is important to 1 Prepare the cell culture insert or the filter membrane 2 Prime the sort line 3 Determine the reference p
53. entrating cells after sorting for 50 mL conical tubes Phosphate buffered saline PBS Dul N A for sorting applications becco s Bovine serum albumin BSA for coating collection tubes 50 mL polypropylene conical screw top to collect sorted sample tubes Cell Concentrator Module option 34013180 for concentrating cells during sorting a BDIS part number unless otherwise noted or not applicable N A 166 FACSCalibur System User s Guide For the Cell Concentrator Module Option a BDIS part number unless otherwise noted or not applicable N A b Available through your local labware distributor 167 Appendix A Consumables and Service Information For the FACSCalibur Instrument Bal seal BDIS 88 20085 00 Spare sheath and waste reservoir 05 10084 00 Sample O ring 88 20014 00 Air filter 41 10020 00 Consumable kit 12 00299 00 a BDIS part number unless otherwise noted or not applicable N A A 2 Service BDIS Customer Support Center 2350 Qume Drive San Jose California 95131 1807 USA Customer Support Center 800 448 2347 BDIS Customers outside the US Contact your local Becton Dickinson representative or distributor BDIS Consumable Parts Order 110 Forbes Boulevard Mansfield Massachusetts 02048 1145 800 448 2347 BDIS 168 Appendix B FACSCalibur Specifications Appendix B FACSCalibur Specifications 170 FACSCalibur System User s Guide CYTOMETER SPECIFICA
54. ents in time As illustrated in Figure 4 3 a cell passes through the red laser beam and then a few microseconds later through the blue laser beam The red excited signal FL4 is electronically delayed so that its signal arrives at the analysis electronics at the same time as all of the blue excited signals FSC SSC FL1 FL2 and FL3 FL3 and FL4 signals are detected with separate PMTs blue laser 488 red laser 635 time delay red excited signal time Figure 4 3 Signal generation in time The Time Delay Calibration electronics finds how long it takes for the cells to travel between beams and sets the time delay to be equal to this time This results in the pulses arriving at the electronics simultaneously ensuring that all parameters for an event are processed together 4 3 4 4 FACSCalibur System User s Guide Dual Threshold You can use the FL4 option to set a threshold for up to two parameters at a time An event must have values above the threshold for both of these parameters before it is considered for analysis When acquiring samples for DNA content analysis for example it is possible to set a threshold on DNA content usually FL2 and also on light scatter Debris particles with low light scatter but high fluorescence would then be rejected and the resulting files would have a more consistent number of cellular events for histogram modeling The use of two thresholds dual thresholding can sometime
55. et the fluid control to RUN 5 Install the sample tube onto the SIP 6 Click Acquire in the Acquisition Control window 118 FACSCalibur System User s Guide When the insert is filled half way with fluid press the air pressure button to pressurize the concentrator vessel Figure 6 2 The LCD displays a value between 000 and 999 Q Adjust the pressure to the predetermined reference value by turning the air pressure adjustment knob Figure 6 2 The fluid should remain at a constant half full level If necessary make minor adjustments to maintain this level Minor pressure adjustments may be necessary for each cell culture insert even after the reference pressure is determined A CAUTION Do not attempt to sort more than 50 000 cells into a 12 mm cell culture insert or 200 000 cells into a 25 mm cell culture insert Sorting a larger number of cells than the insert can accommodate may result in a clogged insert 9 When sorting is complete press Pause and then Abort l Allow the pressure to remain on until the desired amount of fluid remains in the insert 119 Chapter 6 Cell Concentrator Module Option l l Turn off the pressure by pressing the air pressure button The amount of fluid remaining may be based on the desired volume or cell concentration l Remove cells from the insert for further processing or reanalysis BDIS has not optimized and therefore does not support techniques for using the Cell Concentra
56. fa nontarget cell is also in the envelope If another target cell is located just outside the envelope the catcher tube stays in the stream for a longer period of time to capture it The result is high yield capturing as many target cells as possible with less emphasis on purity Exclusion In Exclusion mode a sort occurs when a target cell is identified and there are no nontarget cells in the sort envelope Also if a second target cell is located just outside the sort envelope no special attempt is made to capture this additional target cell The result is high purity and yield that falls between Single Cell and Recovery Sort performance can be optimized by properly adjusting the cell concentration in your sample To do this it is important to understand the relationship between the event rate and the sort rate Figure 5 3 illustrates this relationship when the sort mode is Single Cell Notice that the maximum capture rate for any given concentration of target cells occurs at an event rate of approximately 2000 cells sec An event rate greater than this would result in a gradual decrease in the number of target cells sorted Obtaining 2000 cells sec at low flow 12 uL min needs an input concentration of 10 cells mL Because of variation in flow rate and because some events may be seen by the flow cytometer but not by a hemacytometer it may be necessary to make some adjustment around 10 cells mL FACSCalibur System User s Gu
57. fiers 32 compensation 33 48 51 detectors 45 48 files 28 optimizing 35 50 f figure saving 51 52 threshold 59 instrument setup 35 51 j k laser red diode 7 56 62 argon ion 11 24 55 safety x Lin 32 44 list mode data files 27 Loader FACS 7 LoaderManager 7 Log 32 m Macintosh 5 Basics Tutorial 25 maintenance instrument monthly 135 138 periodic 139 menu Cytometer 40 mirrors dichroic 24 ModFIT LT software 5 n Next Data File 160 164 example 161 163 specifying file increment 163 164 normalization histogram 180 O optical system 23f components 23 24 optics FL4 55 56f optimization 35 50 FACSCalibur System User s Guide options Cell Concentrator Module 105f FACS Loader 7 FL4 55 56 Sorting 118 120 O ring 114 115 141 overlap spectral 33 34f 57f overlapping emissions 41 p PAINT A GATE 8 parameter DDM 63 parameters choosing 39 pasteur pipette 93 PE phycoerythrin 33 PerCP 50 photomultiplier tube PMT 24 32 phycoerythrin PE 33 PMT photomultiplier tube 24 32 power switch 12 PowerPC 6 priming sort line 82 84 112 113 pulse processing 63 q Quadra 650 6 quadrant marker 47 Quadrant Marker tool 47 r Recovery see sort mode red diode laser 7 56 turningon 62 reference pressure 113 114 117 reports 28 S safety biological x electrical x 179 Index 180 saline filter port 124f sample injection port SIP 13f 134f sample injection tub
58. first collection port A i sort line purge button ji ic ee Press the sort line purge button located inside the FACSCalibur collection station Once the button is pressed the valve remains open for approximately 30 seconds You should see fluid dripping into the 50 mL tube _ NOTE If you do not see fluid dripping into the 50 mL tube after you press the sort line purge button see Chapter 8 Troubleshooting before proceeding 83 Chapter 5 Sorting Option Remove the 50 mL tube from the first collection port and place it in the middle collection port 5 Repeat step 3 Remove the 50 mL tube from the middle collection port and place it in the third collection port on the right 7 Repeat step 3 8 Remove the 50 mL tube 9 Place the cytometer in standby FACSCalibur System User s Guide 5 2 Preparing Collection Tubes Collection tubes must be coated with BSA to help maintain cell integrity and increase cell yield during centrifugation Prepare collection tubes at least one hour before you are ready to sort l Fill one to three 50 mL conical tubes with a 4 BSA solution Dilute BSA in 1X PBS 0 1 NaN Pi Place the tubes on ice or in the refrigerator for at least 1 hour Pour the 4 BSA solution from the tubes into a bulk container when the coating process is finished Four per cent BSA solution may be recycled for 1 month Store it at 2 to 8 C A Install the colle
59. fluorescent emission from each laser intercept is imaged at spatially separated positions This permits fluorescence signals to be detected free from cross contamination from the other beam 55 Chapter 4 FL4 Option The diode laser is mounted at right angles to the 488 nm laser Figure 4 1 The beam combiner reflects the red beam and passes the blue beam resulting in two parallel beams that are focused by a common lens The red beam intercepts the sample stream below the blue beam FL 530430 F ssc 488 10 Fl2 585 42 90 10 beam splitter FLA 661 16 7 DM 560SP DM 640LP P half mirror 670LP fluorescence collection lens FL3 beam combiner A FSC diode 488 10 red diode laser focusing lens blue laser 635 nm 488 nm Figure 4 1 FL4 optics 56 FACSCalibur System User s Guide The FL3 signal passes under the half mirror and through a longpass 670 nm filter to the FL3 PMT The FL4 signal is reflected by a half mirror and passes through a bandpass 661 16 nm filter to the FL4 PMT These filters are optimized for simultaneous detection of PerCP and APC Figure 4 2 but other fluorochromes may be used FL1 530 30 FL2 585 42 FL4 661 16 FL8 670 FITC Figure 4 2 Spectral overlap FL1 FL2 FL3 FL4 I7 Chapter 4 FL4 Option 58 4 2 Time Delay Electronics The spatial separation of the beams results in a single particle generating signals at different mom
60. generated by particles passing through the laser beam in the flow cytometer These light signals are converted to electronic signals voltages and 31 Chapter 3 Instrument Setup for Acquisition of Samples then assigned a channel number on a data plot By adjusting the detectors and amplifiers you control where these signals appear on the dot plot PI FSC PZ O SSC 150 1 00 3 PS FL 150 1 00 3 P4 FL 150 1 00 3 PS FL3 150 1 00 Figure 3 1 Detectors Amps window Detectors Voltages Detectors allow you to set the photodiode setting for forward scatter FSC and the photomultiplier tube PMT voltages for SSC FL1 FL2 and FL3 Because the low angle scattering signal is much more intense than other signals a photodiode rather than the more sensitive PMT is used in FSC Amplifiers Amplifiers allow you to make fine adjustments to the signals The Amplifier Mode Lin or Log and Amp Gain allow you to adjust amplifier settings for FSC SSC FL1 FL2 and FL3 32 FACSCalibur System User s Guide Threshold The Threshold window allows you to set a channel number below which data will not be processed Only signals with an intensity greater than or equal to the threshold channel number will be processed by the cytometer Primary Secondary Yalue Param Param s2 FSC H s2 O SSC H SSC H s2 O FLI H FL1 H s2 O FL2 H FL2 H 3 FL3 H 52 i Hone _ NOTE A secondary threshold is ava
61. h filter See Section 2 2 3 step 2 Air bubble in flow cell Press PRIME to drain and fill the flow cell See Section 2 2 3 step 2 Improper sheath pressure Ensure sheath reservoir cap is tight and all connectors are secure Check sheath voltage in Status window Check also for a cracked sheath reservoir 162 Appendix A Consumables and Service Information Appendix A Consumables and Service Information 164 FACSCalibur System User s Guide A l Supplies Required Flow Cytometry Applications 12 x 75 mm Falcon capped 2058 for introducing samples to the polystyrene test tubes FACSCalibur flow cytometer FACSFlow sheath fluid 340398 US balanced electrolyte solution for use as 342003 Europe sheath fluid CaliRBRITE beads 349502 for SSC FL1 FL2 setup and quality control FL3 CaliBRITE beads 340370 for FL3 setup and quality control APC beads for FL4 setup and quality control DNA QC Particles Kit 349523 for setup and monitoring of instrument performance in DNA analysis Chlorine bleach for instrument cleaning a BDIS part number unless otherwise noted or not applicable N A b TBD To be determined c Clorox and other brand name bleaches are preferred because they are filtered to remove particles and contain a known con centration of 5 sodium hypochlorite 165 Appendix A Consumables and Service Information For the Sorting Option Centrifuge with swinging bucket rotor N A for conc
62. he Sort Counters window to select counters to monitor both sorted and aborted cells The Sort Counters window pop up menus display a rate or an accumulation of four values Threshold Auxiliary Sort and Abort l Choose Sort Counters from the Cytometer menu The Sort Counters window appears Threshold Rate 99112 Sort Rate Y 97160 Abort Rate 94705 2 Use the pop up menu for each of the fields in the window e Threshold Rate Threshold Total displays the rate events sec or the total number of cells triggering the threshold These cells are considered for acquisition and sorting including the aborted cells 90 FACSCalibur System User s Guide e Auxiliary Rate Auxiliary Total displays the rate events sec or the total number of cells the FACSCalibur system is processing includes the aborted cells e Sort Rate Sort Total displays the rate events sec or the total number of cells that are sorted e Abort Rate Abort Total displays the rate events sec or the total number of cells meeting the abort criteria Abort characteristics are determined before the electronics decides whether the cell is a target cell 5 6 Sorting the Sample Before beginning a sort be sure e the sheath reservoir is filled with 1X PBS e the sort line is primed with 1X PBS e the BSA coated collection tubes have been installed If you have the Cell Concentrator Module option read Chapter 6 of this user s guide before sorting
63. icolor analysis classification studies of chromosomes DNA content analysis platelet studies and investigation of intracellular ionized calcium measurements 1 2 Components of the Basic FACSCalibur System Hardware e Sensor Unit providing up to three color multiparameter analysis e FACStation data management system including a Macintosh computer monitor 17 or 20 inch and color printer Other computer systems can also be supported for off line data analysis contact your Becton Dickinson Sales Representative for detailed information FACSCalibur System User s Guide Software The FACStation system comes with the following software installed e Macintosh system software version 7 5 3 or later e CELLQuest software version 3 0 or later for acquisition and analysis e FACSComp software version 3 0 or later for instrument setup and quality control e FACSConvert software version 1 0 or later for analyzing Hewlett Packard CONSORT generated data e ModFit Z7 software version 1 0 or later for DNA analysis _ NOTE See Appendix A Consumables and Service Information for a list of 1 3 operating supplies necessary for using the FACSCalibur system See Section 1 4 for application specific software options available from BDIS Installation Your Becton Dickinson Field Service Representative will install and set up your FACSCalibur system CELLQuest FACSComp ModFIT LT and FACSConvert softwar
64. id of excess fluid in the insert Once the excess fluid has been removed turn off the air pressure Be careful not to remove too much fluid Drying out the culture insert will affect cell recovery 121 Chapter 6 Cell Concentrator Module Option Removing Cells for Re analysis l Open the concentrator vessel Unscrew the top of the vessel by turning counterclockwise 2 Gently pipette the fluid up and down to resuspend the cells Avoid creating air bubbles If necessary add PBS to the insert to increase the fluid volume 3 Suction the cell suspension into the pipette and transfer to an appropriate container Cleaning the Sort Line After sorting is completed clean the Cell Concentrator Module sort line Turn off the Cell Concentrator Module use the 60 mL syringe provided and follow these steps 122 FACSCalibur System User s Guide O WARNING If working with biohazardous material perform the cleaning procedure described below first with 1 10 bleach solution followed by twice the volume of distilled water l Disconnect the tubing from the syringe by twisting the luer end counterclockwise 2 Fill the syringe with distilled water Place the syringe nozzle in a container filled with distilled water and slowly pull out the plunger of the syringe until the syringe is full 3 Bleed any air out of the syringe by holding it luer end up and gently pushing in the plunger 4 Reconnect the tubing to the syringe by turning
65. ide 1000 Target cell capture above 300 cells sec not possible 100 mth Sort Rate cells sec 01 100 1000 10000 Event Rate cells sec Multiply sort rate by 12 to get yield cells mL Figure 5 3 Sort yield at various event rates and sample concentration 8l Chapter 5 Sorting Option The FACSCalibur system with the sorting option requires little preparation for sorting Once you have set up for acquisition simply perform the following steps 1 Fill the sheath reservoir with 1X phosphate buffered saline PBS and prime the sort line Other sheath fluids may have a negative impact on the viability of sorted cells 2 Install bovine serum albumin BSA coated collection tubes 1 to 3 tubes or prepare the optional Cell Concentrator Module 3 Identify the population by setting a gate to identify it 4 Define the sort mode and number of cells to be sorted _ NOTE If you are not using the FACSCalibur system for sorting applications follow the maintenance procedure outlined in Section 5 9 Cleaning the Sort Line to fill the sort line with distilled water This prevents the accumulation of saline deposits in the line 5 1 Priming the Sort Line Prime the sort line to ensure that the sort lines are clog free Install a tube of distilled water on the FACSCalibur instrument while in RUN mode 82 FACSCalibur System User s Guide 2 Install a 50 mL tube in the first collection port on the left
66. ide four log decade range Fluorescence spectral overlap can be compensated between FL1 and F12 between FL2 and FL3 channels and between FL3 and FL4 with FL4 option Width and Area measurements for discriminating doublets available for all fluorescence parameters Time available correlated to any parameter for kinetic experiments or other applications SAMPLE LOADING SPECIFICATIONS Tube lifter design with multiple sensors which verify rack identification and tube position 40 12 x 75 mm tubes per rack Up to 16 racks per FACS Loader Data Entry Loader Control Barcode Scanner optional Mixing Mode Sorting Purity Capture Rate Sort Modes Recovery Sterile Sorting Workstation Central Processing Unit CPU Memory Level 2 Cache Data Storage Networking CD ROM Monitor Data File Structure FACSCalibur System User s Guide Sample information reagent panels and rack information can be defined for up to 640 tubes 40 tubes x 16 racks at a time Automated control through WorklistManager software and manual control with stat interrupt capability through FACS Loader electronic keypad Automates data entry for Codabar Code 39 Interleaved 2 of 5 Code 2 of 5 and Code 128 Adjustable High Energy and Low Energy mix SORTING SPECIFICATIONS gt 95 300 cells second Three modes all aerosol free single cell exclusion and recovery Depends upon sample and sorting conditions gt 50 System design
67. igure 6 2 Begin by turning the knob to approximately 500 Gradually adjust the pressure while monitoring how the changes affect the fluid level in the insert The fluid should remain at a constant half full level Increasing the pressure increases the rate at which fluid flows through the insert Decreasing the pressure decreases the rate at which fluid flows through the insert If fluid is rapidly filling the insert click PAUSE to temporarily stop acquisition and sorting Turn the knob clockwise to increase the pressure while allowing the fluid to decrease Resume sorting If the fluid level is rapidly decreasing press the air pressure button to turn off the pressure Turn the knob counterclockwise to decrease the pressure while allowing the insert to fill with fluid Do not forget to turn the pressure on again to avoid overfilling the concentrator vessel Once the reference pressure has been established click Pause and then Abort l 2 Turn off the pressure to the vessel by pressing the air pressure button 1 3 Set the fluid control to STNDBY 117 Chapter 6 Cell Concentrator Module Option Sorting and Concentrating Cells l Add 100 mL of undiluted bleach to the empty waste reservoir before sorting into the concentrator vessel 2 Make sure the Cell Concentrator Module is on and the concentrator vessel is properly assembled with the cell culture insert properly installed 3 Make sure the LO flow rate is selected 4 S
68. ilable only with the FL4 option Changing the secondary threshold selection will have no effect on instruments that do not have the FL4 option Compensation Fluorochromes emit light over a range of wavelengths therefore a signal from one fluorochrome may overlap in a detector used for another fluorochrome For example fluorescein FITC appears primarily in the FL1 detector but some of its fluorescence overlaps into the FL2 detector Phycoerythrin PE appears primarily in the FL2 detector but some of its fluorescence overlaps into the FL1 and the FL3 detectors Figure 3 2 illustrates this 33 Chapter 3 Instrument Setup for Acquisition of Samples FL1 530 30 FL2 585 42 FL3 650 FITC 500 600 700 Figure 3 2 Spectral overlap FL1 FL2 FL3 The Compensation window allows you to adjust for this spectral overlap when the samples are stained with two or more fluorochromes You will practice adjusting compensation in Section 3 2 FLI 0 0 3 FL FL2 o o FLI FL2 a 0 3 FL3 FL3 o o FL FL3 6 001 FL4 FL4 6 67 FL3 34 FACSCalibur System User s Guide 3 2 Optimizing the Instrument Settings Optimization is the instrument adjustment procedure that sets the detectors amplifiers threshold and compensation for specific samples When you install a tube on the cytometer you can view a display of the data and make any necessary adjustments before acquiring the sample The optimization
69. ill go Retighten the retainer 148 CHAPTER 8 Troubleshooting CHAPTER 8 Summary E symptoms you might observe during instrument operation possible causes and solutions 150 SYMPTOM FACSCalibur System User s Guide The following is a list of symptoms their possible causes and solutions For technical information not covered in this troubleshooting guide contact Becton Dickinson Immunocytometry Systems Customer Support Center at 800 448 2347 BDIS DATA DISPLAY No events in acquisition display If the Status window reads READY check the following Cause Solution Cause Solution Cause Solution Cause Solution Cause Solution Cause Solution Cause Solution Threshold parameter gain setting too low Increase threshold parameter gain setting See Section 3 1 Threshold level too high Lower threshold level Threshold not set to correct parameter usually FSC Set threshold to correct parameter appropriate to application No sample in tube Add sample to tube or install new sample tube Sample not mixed properly Mix sample to suspend cells Sample injection tube clogged Remove sample tube to allow back flushing If clog persists clean sample injection tube See Section x Gate set with no data passing through gate Delete gate Refer to the CELL Quest Software Users Guide 151 Chapter 8 Troubleshooting 152 Cause Solution Cau
70. in the filter gently tap the filter body to dislodge them and force them to the top Push the roller in the pinchcock forward to allow the pressurized sheath fluid to force the air bubbles into the waste reservoir When the air bubbles are expelled return the roller to its original position to stop the flow of sheath to the waste reservoir FACSCalibur System User s Guide l 4 Return roller in the pinchcock to its original position once the filter is filled with fluid l 5 Record the replacement date on the outside of the filter Cleaning the Air Filter The air filter located above the fluid reservoirs cleans the air that cools the FACSCalibur laser The filter can be vacuumed or washed and air dried Check the filter periodically but clean it only if it is dirty l Remove the filter by grasping the edges and pulling gently to slide it out 143 Chapter 7 Cleaning and Maintenance 144 Vacuum the air filter or hold it under running tap water to clean it If the filter is rinsed with water allow it to dry completely before reinstalling Reinstall the filter Be sure the arrows along the edge of the filter are pointing up indicating the direction of air flow Align the right edge of the filter against the spring clips in the slide rail Push forward slowly Changing the Bal Seal
71. ission optical train Four high performance high dynamic range photomultipliers with band pass filters 530 nm FITC 585 nm PE PI 661 nm APC and gt 650 nm PerCP with base unit gt 670 nm PerCP with FL4 option 171 Appendix B FACSCalibur Specifications Fluidics General Operation Fluid Reservoirs Sample Flow Rates Quartz Cuvette Sample Concentration Signal Processing Workstation Resolution Dynamic range Fluorescence Compensation Networks Pulse Processing Time Sample delivery Rack Capacity Rack Support 172 Front key panel control provides three modes RUN STNDBY and PRIME automatic standby mode conserves sheath fluid by stopping sheath flow when no sample tube is installed Easily accessible 4 L capacity sheath and waste containers are housed in a convenient pull out drawer level detectors automatically indicate low levels of sheath or high levels of waste Three selectable flow rates of 60 UL min 35 uL min and 12 uL min Pressure difference between sheath and sample is regulated and monitored particle velocity in flow cell is approximately 6 meters second Internal cross section is rectangular 430 um x 180 um external surfaces are anti reflection coated for maximum transmission of laser light Single cell suspension of 10 to 2 x 10 particles mL recommended range 1024 channels on all parameters Logarithmic amplifiers for SSC FL1 FL2 FL3 and FL4 with FL4 option prov
72. les in sheath filter Push the roller in the pinchcock forward to purge air from the filter Sample not diluted in same fluid as sheath fluid Dilute sample in the same fluid as you are using for sheath If you are running CaliBRITE beads dilute them in FACSFlow and use FACSFlow for sheath fluid Quality control particles are old or contaminated Make new QC solution and try quality control procedure again INSTRUMENT Droplet containment vacuum not functioning Cause Solution Cause Solution Cause Solution Cause Solution O ring in retainer worn Replace O ring See Section 7 2 6 Outer sleeve not seated in the retainer Loosen retainer push outer sleeve up into the retainer until seated Tighten retainer See Figure 7 7 Outer sleeve not on the sample injection tube Replace outer sleeve loosen retainer slide outer sleeve over sample injection tube until it is seated tighten retainer Waste line pinched preventing proper aspiration Check waste line SYMPTOM SYMPTOM SYMPTOM FACSCalibur System User s Guide Flow cell will not fill Cause Solution Cause Solution Cause Solution No sheath pressure 1 Check to see vent valve toggle switch is in correct position 2 Tighten sheath reservoir cap 3 Check to see all sheath fluid connectors are securely seated 4 Check for leak or crack in sheath reservoir Replace reservoir if necessary 5 Check status sam
73. mple e Chapter 6 Cell Concentrator Module Option explains how to sort directly onto filters or cell culture inserts and how to recover sorted cells without centrifugation e Chapter 7 Cleaning and Maintenance provides instructions necessary to clean and maintain your instrument e Chapter 8 Troubleshooting lists some of the problems you may encounter during operation and suggests possible solutions e Appendix A Consumables and Service Information provides a list of consumable parts and their order numbers and phone numbers for order information and technical support e Appendix B FACSCalibur Specifications provides a more detailed description of the instrument Conventions Used in This Guide Italics Bold NOTE A CAUTION Q WARNING Highlights any text that appears on the screen Indicates actions or steps to perform Points out additional information that may be helpful or hints for better or easier operation Alerts you to situations that could result in instrument damage failure in a procedure or possible incorrect data Alerts you to situations that could result in injury Preface Help For technical questions or assistance in solving a problem 1 Read the section of the manual specific to the instrument operation that you are performing Use the table of contents and index to locate this information 2 See Chapter 7 for troubleshooting information 3 US customers call the Becton
74. multiparameter capability There are various options and upgrades available for your particular needs The FL4 option equips the FACSCalibur system with a second laser red diode that intercepts the sample stream in a spatially separated location to provide a fourth fluorescence parameter This red diode laser offers additional flexibility in fluorochrome choice for multicolor research analysis The FACS Loader provides automated introduction of prepared samples to the FACSCalibur flow cytometer The FACS Loader features removable 40 tube carousels on board mixing LoaderManager and WorklistManager software for programming acquisition of up to 640 tubes The Sorting option is useful for sorting cells for verification of morphology or molecular studies or for sorting viable cells that can be returned to culture or used in functional assays All sorting applications are for research use only The Cell Concentrator Module collects sorted cells and removes excess sheath fluid resulting in a more concentrated sample for further processing or analysis BDIS has not optimized and therefore does not support techniques for using the Cell Concentrator Module to recover viable cells i Chapter 1 Introduction FACStation Software The following application specific software programs are available from BDIS for use with the FACSCalibur system e SimulSET software for automated acquisition and analysis of two color immunophenotyping e At
75. n the Status window should return to its normal value FACSCalibur System User s Guide Emptying the Waste Reservoir WARNING Blood samples may contain infectious agents hazardous to your health Wear gloves when handling blood or any materials with which it comes in contact Follow local state and federal biohazard waste handling regulations when disposing of biohazardous material Empty the waste reservoir when you fill the sheath reservoir This prevents the waste reservoir from overflowing Keep a spare waste reservoir on hand as a replacement the full reservoir should be allowed to sit for 30 minutes before emptying to disinfect waste fluid l Slide out the fluidics drawer Disconnect the waste tubing orange and the waste air vent tubing white from the FACSCalibur instrument Squeeze the metal clip on the quick disconnects and pull each connector from the fitting 3 Disconnect the waste fluid detection probe cable Squeeze the tabs at the sides of the connector and pull 19 Chapter 2 Getting Started 20 A Remove the waste reservoir O WARNING Wait at least 30 minutes after the completion of the last run before disposing of waste reservoir contents This helps to ensure that biohazardous materials are inactivated before disposal Unscrew the cap assembly from the reservoir and set the assembly aside Empty the reservoir according to local state and federal biohazard waste handling regulations
76. n you shut down the instrument each day clean the sample injection tube and the area between the injection tube and the outer sleeve Figure 7 1 The outer sleeve covers the injection tube and functions as part of the droplet containment system Fluid dripping from the injection tube is aspirated through the space between the tube and outer sleeve Clean the sample injection tube to prevent it from becoming clogged and to remove dyes that can remain in the tubing Perform this procedure before shutdown and immediately after running viscous samples or dyes such as propidium iodide PI acridine orange or thiazole orange 133 Chapter 7 Cleaning and Maintenance outer sleeve sample injection tube tube support arm Figure 7 1 Sample injection port SIP l Move the tube support arm to the side 2 Install a tube containing 3 mL of a 1 10 dilution of bleach on the SIP Allow the vacuum to aspirate 2 mL of the bleach solution 3 Press the HI sample flow rate button and center the tube support arm Allow the bleach to run for 5 minutes 134 FACSCalibur System User s Guide With the tube support arm moved to the right install a tube containing 3 mL of distilled water on the SIP Allow the vacuum to aspirate 2 mL of the water 5 Center the tube support arm and allow the water to run for 5 minutes 6 Press the STNDBY fluid control button If the tube contains more than 1 mL of distilled water remove some water bef
77. nu displaying the available choices Figure 3 6 38 FACSCalibur System User s Guide Dot Plot Plot Source Acquisition Acquisition gt Analysis Pe Selesi Fila Pig Re ts Y Parameter S5C H 1074 Gate Shewi GG ef Bela Figure 3 5 Choosing an acquisition dot plot Dot Plot Plot Source Seteet File Figi x Parameter 1024 Y Parameter 10274 1024 Gate 10274 1024 E Resa a E E 1074 Time 1074 Time 107 40 sec_ Figure 3 6 Choosing parameters 7 Click OK The dot plot appears in the Experiment document 39 Chapter 3 Instrument Setup for Acquisition of Samples 40 gt The next step is to open all the necessary instrument settings windows using the Cytometer menu You will adjust the settings in each window to best view your samples 8 Choose Detectors Amps from the Cytometer menu The Detectors Amps window appears Use this window to adjust the voltages and amplifiers for all the available parameters Pi FSC P2 S9C P FL1 P4 FL PS FL3 9 Choose Threshold from the Cytometer menu The Threshold window appears Figure 3 7 Use this window to select threshold parameter Any particle must have some signal in that parameter for the cytometer to recognize it 10 FACSCalibur System User s Guide Primary secondary Yalue Param Param s2 FSC H s2 O SSC H SSC H s2 O FLI H FL1 H s2 O FL2 H C FL2 H s2 O FL3 H FL3 H s2 E
78. o do this increase the FL3 FL4 compensation value Notice the APC labeled beads move toward the y axis FL4 Continue to adjust until the entire population is to the left of the vertical marker line Figure 4 11 00 1000 wr m T 5 oJ 400 400 B00 800 1000 Figure 4 11 Adjusted FL8 FL4 compensation 3 0 Adjust the FL4 FL3 compensation while viewing the FL3 vs FL4 plot Adjust to rid the FL4 detector of FL3 fluorescence overlap To do this increase the FL4 FL3 compensation value Notice the PerCP labeled beads move toward the x axis FL3 Continue to adjust until the entire population is below the horizontal marker line Figure 4 12 Continued increases in compensation values may not cause the population to move toward the x axis To check that compensation is set correctly make sure that decreasing compensation will cause the population to move above the marker 72 Chapter 4 FL4 Option 74 COMP ADJUSTED 001 800 1000 o So D o Se wT o 5 ol 200 400 B00 300 1000 Figure 4 12 Adjusted FL4 FL3 compensation _ NOTE If you have difficulty achieving the correct compensation levels perform the Time Delay Calibration procedure again You have now completed the instrument adjustments necessary for you to view and analyze four color data You have also performed Time Delay Calibration necessary to ensure that the signals generated from the blue and red lasers arri
79. of the tube loosen the retainer slightly and push the sleeve up as far as it will go Retighten the retainer FACSCalibur System User s Guide Changing the Sample O ring The sample tube O ring located within the retainer forms a seal that allows the droplet containment vacuum to function properly The O ring should be replaced when droplets form at the end of the sample injection tube while the vacuum is operating NOTE Follow good laboratory practices wear gloves whenever cleaning the instrument or replacing parts l Remove the outer droplet sleeve from the sample injection tube Turn the retainer counterclockwise and pull the outer sleeve from the retainer See Figure 7 5 2 Invert the retainer and allow the O ring to fall onto the benchtop If the O ring does not fall out initially tap the retainer on the benchtop to dislodge the O ring 3 Drop the new O ring into the retainer Make sure the O ring is seated properly in the bottom of the retainer 147 Chapter 7 Cleaning and Maintenance A Reinstall the retainer and the outer sleeve Tighten the retainer enough to hold it in place and slide the outer sleeve over the sample injection tube into the opening of the retainer Continue tightening the retainer 5 Install a sample tube on the SIP This ensures 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 w
80. ore turning off the power to the instrument The distilled water should remain on the SIP after the instrument has been turned off to prevent salt deposits from forming in the sample injection tube Monthly Cleaning An overall fluidics cleaning is required to remove debris and contaminants from the sheath tubing waste tubing and flow cell Perform system fluidics cleaning at least once a month or more frequently if you are running a high volume of samples or adhesive dyes such as PI acridine orange or thiazole orange You will need a spare reservoir for this procedure See Appendix A Consumables and Service Information for details 135 Chapter 7 Cleaning and Maintenance 136 l Remove the sheath reservoir See Section 2 2 1 Filling the Sheath Reservoir for instructions on removing the sheath reservoir If the system is pressurized push the vent valve toggle switch in the direction of the arrow to release pressure from the sheath reservoir Remove the sheath filter tubing Disconnect the top white quick disconnect that secures the filter to the instrument by squeezing the metal clip on the quick disconnect and pulling the connector from the fitting Install a spare reservoir containing 1 to 2 L of a 1 10 dilution of bleach Prepare the bleach solution by adding one part undiluted bleach to nine parts distilled water Connect the sheath tubing white from the reservoir to the port labeled Saline Filter Figure 7 2
81. ould see fluid dripping from the sort line into the lower chamber of the concentrator vessel Once the button is pressed the valve remains open for approximately 30 seconds 5 If the sort line is clear set the fluid control to STNDBY If there is a clog massage the sort line and press the sort line purge button again _ NOTE If you do not see fluid dripping into the Concentrator vessel after you press the sort line purge button see Chapter 8 Troubleshooting before proceeding Determining Reference Pressure It is important to determine the reference pressure for each size and type of culture insert or filter membrane you use The pressure is adjusted to maintain a half full level of sheath fluid in the cell culture insert or filter holder Once you have established the target pressure for a particular size insert you can use m LIS Chapter 6 Cell Concentrator Module Option 114 this as the starting point when setting the pressure for other inserts of the same size Minor pressure adjustments may be necessary for each cell culture insert even after the reference pressure is determined l Place a rubber O ring into the retainer in the lower compartment of the vessel 2 Place the cell culture insert holder on top of the O ring See Figure 6 6 3 Place a second O ring on top of the insert holder 4 Make sure the O rings fit in the grooves of the insert holder See Figure 6 6 FACSCalibur System User s Gui
82. ove it from the filter body Save this piece to attach to the new filter Remove the output tubing from the output port by pulling the tubing from the port Save the tubing to attach to the new filter and discard the old filter A CAUTION Droplets of sheath fluid could be ejected as the output tubing is pulled off This would depend on how forcefully the tubing is pulled off Use a paper towel to cover the tubing and output port as the tubing is pulled off 7 Install a new O ring around the threads at the bottom of the new filter Push the O ring as far up to the threads as possible 8 Attach the base from step 5 to the new filter by screwing until snug Attach the output tubing from step 6 to the new filter by pushing the tubing onto the output port 141 Chapter 7 Cleaning and Maintenance 142 10 11 12 13 Install the filter into the fluidics drawer by pushing each quick disconnect firmly into its fitting until you hear a click Replace the filter so that the base is at the bottom and the output port is at the top Reattach the air vent tubing from step 4 to the new filter by screwing the fitting onto the vent port Pressurize the instrument b y pulling the vent valve toggle switch forward Fill the newly installed filter with fluid Push the RUN fluid control button and then push the roller in the pinchcock forward to allow the air to escape as the filter fills with fluid If bubbles are visible
83. ple voltage in RUN with test tube off Sheath reservoir empty Fill sheath reservoir See Section 2 2 1 Air in sheath filter Vent air from sheath filter See Section 2 2 3 step 1 Sample tube does not fit on SIP Cause Solution Cause Solution Sample tube other than Falcon brand is being used Use Falcon brand sample tubes Bal seal is worn and needs to be replaced Replace the Bal seal See Section 7 3 3 FL4 FL3 compensation more than twice usual Cause Solution Sheath reservoir cap not tightened Tighten sheath reservoir cap 161 Chapter 8 Troubleshooting SYMPTOM SYMPTOM Cause Solution Cause Solution Cause Solution Air in sheath filter Vent air from sheath filter See Section 2 2 3 step 2 Air bubble in flow cell Press PRIME to drain and fill the flow cell See Section 2 2 3 step 2 Timing between blue and red laser is inaccurate Perform Time Delay Calibration Time Delay Calibration results in error message stating there is an insufficient signal Cause Solution Incorrect Time Delay Calibration setup See Section 4 4 for proper Time Delay Calibration setup Time Delay Calibration results in error message stating signal is out of tolerance Cause Solution Cause Solution Cause Solution Cause Solution Sheath reservoir cap not tightened Tighten sheath reservoir cap Air in sheath filter Vent air from sheat
84. procedure depends on the application as well as the number of fluorochromes used Typically you will view an FSC vs SSC plot to ensure that all relevant cell populations are on scale for these parameters Additionally if fluorochromes are used you can view fluorescence plots and adjust PMT voltages detector amplification and compensation as necessary In the following exercise you will use CaliBRITE beads to practice adjusting instrument settings for a three color sample acquisition A tube of unstained CaliBRITE beads is used to set detectors amps and threshold and a mixed bead tube containing unstained FITC PE and PerCP beads is used to adjust compensation l Prepare two 12 x 75 mm tubes containing CaliBRITE beads One tube contains unlabeled CaliBRITE beads and the second tube contains a mixture of unlabeled FITC PE and PerCP CaliBRITE beads Refer to the CaliBRITE Beads package insert for instructions 35 Chapter 3 Instrument Setup for Acquisition of Samples 2 Choose CELLQuest from the Apple menu to launch the software The CELLQuest desktop appears displaying an untitled Experiment document File Edit Cytometer Plots Gates Stats Acquire Windows C untitled 1 Menu bar Tool palette Figure 3 3 CELLQuest Experiment document window Alternately you can start the program by double clicking the program icon located in the BD Applications folder on the computer hard drive Refer to the CELL
85. r System User s Guide If all tubes on the collection station fill before any of these conditions is met sorting continues but the sorted sample is sent to the waste reservoir To continue sorting after the collection tubes are filled click Pause replace the collection tubes with clean BSA coated tubes and click Resume 5 8 Recovering Sorted Cells Because the sorted sample is dilute it is necessary to concentrate it before proceeding to analysis l Remove the collections tube s from the instrument Cap each tube once you have removed it 2 Spin the tubes at 300 x g for 5 minutes A longer spin may improve recovery for some cell types 3 Aspirate the supernatant by using a pasteur pipette and a vacuum system Be careful not to disturb the pellet 95 Chapter 5 Sorting Option 5 9 4 Resuspend the pellet with 100 uL of PBS Cleaning the Sort Line You should flush the sort line periodically to remove cell debris and saline deposits Flushing is especially necessary if there is a reduction in the amount of fluid entering the collection tubes during a sort For this procedure use the 60 cc syringe provided If working with biohazardous material perform the following cleaning procedure first with 1 10 bleach solution followed by twice the volume of distilled water If sorting will not be performed for an extended period of time 2 to 3 weeks follow this procedure to fill the sort line with distilled water This
86. r cracks Bal seal worn Replace Bal seal See Section 7 2 5 Sheath reservoir bracket not depressing pressure button under bracket Tighten screw on top right hand side If the Status window reads NOT READY check the following Cause Solution Cause Solution Cause Solution Laser warming up Wait 5 minutes Laser not functioning Check laser power in the Status window If power is 0 mWatts turn off the FACSCalibur instrument and computer then turn on instrument followed by the computer If power is still 0 mWatts contact BDIS Leak at sheath area Check Status window with test tube off sample injection port and instrument in RUN mode Sample voltage should be 10 2 approximate If under 10 0 replace sheath reservoir then replace cap and finally replace gasket 153 Chapter 8 Troubleshooting SYMPTOM 154 Cause Solution Cause Solution Sheath reservoir empty or waste reservoir full Check reservoirs fill sheath and empty waste if necessary See Section 2 2 1 and Section 2 2 2 Electronic connector for the sheath or waste reservoir is not properly seated in the connector port Firmly seat the connector into its port High sample event rate Cause Solution Cause Solution Cause Solution Cause Solution Cause Solution Cause Solution Air bubble in flow cell Press PRIME to drain and fill the flow cell See Section 2 2 3 step 2 Ai
87. r in sheath filter Vent air from sheath filter See Section 2 2 3x step 2 Threshold level too low Increase threshold level Threshold parameter gain setting too high Decrease threshold parameter gain setting Sample too concentrated Dilute sample Cell concentration should be 1 x 10 to 1 x 10 cells mL for optimal event rates Sample flow rate set on HI Set sample flow rate to MED or LO SYMPTOM SYMPTOM FACSCalibur System User s Guide Low sample event rate Cause Solution Cause Solution Cause Solution Cause Solution Cause Solution Threshold level too high Lower threshold level Threshold parameter gain setting too low Increase threshold parameter gain setting Sample not adequately mixed Mix sample to suspend cells Sample too dilute Concentrate sample If flow rate setting is not critical to the application set flow rate switch to MED or HI Clog in sample injection tube Run 10 bleach and warm deionized water for 20 minutes followed by deionized water for 10 minutes Erratic event rate Cause Solution Cause Solution Cause Solution Sample tube cracked Replace sample tube Bal seal worn Replace Bal seal See Section 7 2 5 Partially blocked sample injection tube Remove sample tube to allow back flushing If event rate is still erratic clean sample injection tube 155 Chapter 8 Troubleshooting SYMPTOM SYMPTOM 156 C
88. ressure Cell culture inserts should be coated with a bovine serum albumin BSA solution before use This prevents sorted cells from sticking to the inserts and facilitates the removal of cells from the inserts when sorting is complete A filter membrane may not need to be coated with BSA prior to use However you must assemble the filter membrane and filter membrane holder before sorting Follow the steps below for the type of insert you are using CAUTION Cell culture inserts and filters with a pore size smaller than 1 Um may result in a concentration rate that is lower than the rate at which fluid enters the insert This may cause overflowing of fluid from the culture insert or filter holder while sorting Preparing the cell culture insert l Place a cell culture insert in a Multiwell tissue culture plate 2 Fill the cell culture insert and the well with a filtered 4 BSA solution Dissolve the 4 BSA solution in 1X PBS 0 1 NaN 109 Chapter 6 Cell Concentrator Module Option 110 3 Cover the tissue culture plate and store overnight in the refrigerator Inserts may be stored at 4 C for up to 1 month 4 Just before using the insert rinse it with 1X PBS Pour out the BSA solution Flush the tissue culture well and fill with PBS Pour out the PBS Use the insert immediately Preparing the filter membrane l Place an individual filter membrane over the bottom of the filter holder Use a pair of filter forceps It m
89. rument settings are already set proceed to step 8 l Launch CELLQuest software See Section 3 1 Accessing Instrument Controls in CELLQuest and Section 3 2 Optimizing Instrument Settings for information on using CELLQuest software Refer to the CELL Quest Software Users Guide for specific instructions Choose Connect to Cytometer from the Acquire menu Acquire Acquisition amp Storage Parameter Description Counters Edit Reagent List Edit Panels Sort Region Selection FACSCalibur System User s Guide 3 Choose Instrument Settings from the Cytometer menu The Instrument Settings dialog box appears Instrument Settings Cytometer Type FACSCal ibur Detectors fisps Param Detector Voltage AspGain E00 1 00 Displaying Current Status 4 Click Open A standard location dialog box appears Navigate to the folder where you saved the instrument settings file from the exercise in Section 3 3 5 Select the file and Click Open The dialog box disappears and the saved instrument settings appear in the Instrument Settings window 61 Chapter 4 FL4 Option 6 Click Set The instrument settings are sent to the FACSCalibur flow cytometer 7 Click Done The Instrument Settings window disappears gt The next step is to turn on the red diode laser 8 Choose Detectors Amps from the Cytometer menu Click in the Four color checkbox to turn on the red diode laser Notice that P7 changes to FL
90. s be imitated by using an acquisition gate However when the event rate with a single threshold remains too high for proper acquisition either because of a high abort rate or a data rate too high for computer acquisition dual thresholding can be the best solution Because of the difference in detector and processing electronics between FSC and the other channels some care should be taken when using FSC in dual thresholding Make sure signals in other channels appear as expected after the FSC threshold level is set BDIS does not recommend setting a FSC threshold that would split a population of cells or beads Setting Up the FACSCalibur Instrument for Four Color Analysis In this section you will learn how to turn on the red diode laser perform Time Delay Calibration and adjust the detector amp and compensation settings for the FL4 parameter 59 Chapter 4 FL4 Option 60 You will use APC beads to set the FL4 detector and amplifier and PerCP beads and APC beads to set compensation for the FL4 parameter Make sure you have performed the set up procedure in Section 3 2 before you begin If you previously performed the exercises in Section 3 2 Optimizing the Instrument Settings and Section 3 3 Saving Instrument Settings you set and saved instrument settings for FL1 FL2 and FL3 parameters Use CELLQuest software to retrieve them for use in the following exercise If you just completed the exercise in Section 3 2 and the inst
91. se Solution Cause Solution Cause Solution Using an analysis plot Reformat plot to acquisition Bal seal worn Replace Bal seal See Section 7 2 5 Communication failure between computer and FACSCalibur instrument Turn off computer and FACSCalibur instrument Turn on instrument followed by computer GPIO error cannot read instrument status Turn off computer and FACSCalibur instrument Reseat GPIO cable located next to power cord in back of cytometer Repower If the Status window reads STNDBY check the following Cause Solution Cause Solution Cause Solution Cause Solution Cause Solution The RUN fluid control button is not activated Press the RUN fluid control button Sample tube not installed or not properly seated Install sample tube on the cytometer Sample tube cracked Replace sample tube Sheath reservoir cap not tightened Tighten sheath reservoir cap Sheath reservoir bracket not replaced Install the bracket See Section 2 2 1 step 10 Cause Solution Cause Solution Cause Solution Cause Solution FACSCalibur System User s Guide Vent valve toggle switch pushed away from you sheath reservoir is vented Flip toggle switch toward you to pressurize the reservoir Sheath reservoir tubing or sheath filter tubing not properly connected Check that all tubing connectors are securely seated Check sheath reservoir fo
92. t The sample injection port SIP is the area on the instrument where the sample 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 backflows from the injection tube Bal seal outer sleeve F sample injection tube tube stop i tube support arm Figure 2 3 Sample injection port SIP 13 Chapter 2 Getting Started e Sample injection tube Stainless steel tube that carries cells from the sample tube to the flow cell this tube is covered with an outer sleeve that serves as part of a droplet containment system 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 2 2 Fluidics Drawer Components Take a few minutes to study Figure 2 4 to become familiar with the fluidics drawer components vent valve toggle switch metal bracket fluid detection probe cables ball valve air supply tubing waste tubing waste air vent tubing sheath tubing waste reservoir Steal UMEE R OE sheath filter air vent tubing sheath reservoir sheath filter Figure 2 4 Fluidics drawer FACSCalibur System User s Guide The fluidics drawer se
93. t see Chapter 8 Troubleshooting before proceeding FACSCalibur System User s Guide l 4 Click Acquire in the Acquisition Control window Events appear in the dot plot Since the Setup box is checked in the Acquisition Control window you can click Acquire and view real time acquisition display without saving the data to a file gt The next step is to adjust the forward scatter amplifier to ensure the CaliBRITE bead signal is above the threshold l 5 Adjust the FSC Amp Gain to 2 0 in the Detectors Amps window This should be high enough to ensure CaliBRITE beads are detected Since the side scatter voltage has not been adjusted all the events are along the forward scatter axis of the plot and low in side scatter Figure 3 8 Beads 001 B00 71000 SSH yo T Caas 700 400 p Figure 3 8 Adjusted FSC The Counters window indicates the rate that the beads are detected by the cytometer 43 Chapter 3 Instrument Setup for Acquisition of Samples 44 l 6 Adjust the SSC PMT Voltage using the Detectors Amps window Click the up or down arrow for the detector level or click the icon between the arrows to display a slider and drag to the appropriate value Place the bead population in the middle of the side scatter range Figure 3 9 The light signals are multiplied by applying a voltage between 150 and 999 to the PMT As the voltage is increased the signal increases and the data appears
94. tation data management system 29 Chapter 2 Getting Started The following hardware and software are included with the FACStation data management system Hardware e Macintosh computer e 17 or 20 inch color monitor e Keyboard e Mouse e Printer color or black and white e Security module Software For detailed information on any of the following software programs installed on the FACStation computer refer to the appropriate software user s guide e Apple Operating System 7 5 software or later e FACSComp software instrument setup and performance evaluation program that assists in setting up the FACSCalibur instrument for immunophenotyping e CELLQuest software provides an easy to use mouse driven interface with pull down menus and windows that display data in a variety of plots including histograms dot plots contour plots and density plots In addition CELLQuest offers acquisition with real time statistics various tools for data analysis instrument control and data storage capabilities e ModFit LT software assists with automatic DNA analysis of files collected with CELLQuest software 26 FACSCalibur System User s Guide FACSConvert software converts CONSORT generated computer files Hewlett Packard HP from the Flow Cytometry Standard FCS 1 0 format to the current FCS 2 0 file format necessary for all FACStation software NOTE To analyze CONSORT generated files you will also need a fil
95. ted parameter is assigned to P6 FL4 height FL4 H is assigned to P7 If you select FL4 the area is assigned to P6 and FL4 width FL4 W is assigned to P7 The following table illustrates your available parameter choices with the red laser on DDM Parameter Parameter 6 P6 Parameter 7 P7 FEI FL1 A FL4 H FE2 FL2 A FL4 H FL3 FL3 A FL4 H FL4 FL4 A FL4 W a area gt The next step is to perform Time Delay Calibration The Time Delay Calibration electronics synchronizes the FSC signal and the FL4 signal in time BDIS recommends performing Time Delay Calibration as part of daily FACSCalibur instrument setup Changes in sheath flow rate might change the number of microseconds it takes a particle to go from the red beam to the blue beam To synchronize the FSC signal and the FL4 signal in time 9 Select Open from the CELLQuest File menu A standard dialog box appears Figure 4 6 64 10 11 FACSCalibur System User s Guide CELLQuest Folder Macintosh HD Fi Time Delay Calibration Desktop Figure 4 6 Standard dialog box Navigate to the Time Delay Calibration document Select the file and click Open If this document is not already in a folder on your hard drive you can find it on the diskette that came with this user s guide Make sure you copy the document onto your hard disk for future use Notice the Time Delay Calibration document Figure 4 7 contains two acquisition histogram plots one
96. tor Module to recover viable cells Recovering Sorted Cells from the Sort Line Some cells may remain in the sort line and not make it to the concentrator vessel and insert before sorting ends This is referred to as dead sort volume You can collect these cells or you may wish to clear them from the sort line to prepare for another sort l When sorting ends remove the sample tube from the cytometer If you want to collect the cells remaining in the sort line leave the insert in the concentrator vessel Remove any collection tubes from the first two collection ports This ensures that when the purge button is pushed the cells are collected in the insert and not in the collection tubes 120 FACSCalibur System User s Guide Install a tube of PBS on the SIP and make sure the fluid control is set to RUN Make sure the fluid level in the insert is low enough to accommodate additional fluid entering the insert once the sort line purge button is depressed If the fluid volume is not low enough turn on the air pressure to the Cell Concentrator Module chamber by depressing the air pressure button This will get rid of excess fluid from the insert Turn off the air pressure once the desired fluid level is reached Push the sort line purge button located in the collection station Once the purging is finished turn on the air pressure to the Cell Concentrator Module chamber vessel by depressing the air pressure button This gets r
97. tractors software for innovative hierarchical data analysis automation e PAINT A GATE software for exploratory multidimensional data analysis and automation I CHAPTER 2 Getting Started CHAPTER 2 Summary FACSCalibur instrument overview fluidics system components optical system components electronics system FACStation data management system overview 2 1 FACSCalibur System User s Guide FACSCalibur Instrument Overview The FACSCalibur standard instrument configuration is a five detector flow cytometer that consists of fluidic optical and electronic systems and a built in air cooled argon ion laser The FACSCalibur system consists of a sensor unit the FACStation data management system and various software packages Sensor Unit As illustrated in Figure 2 1 the basic FACSCalibur sensor unit houses the power switch the fluid control panel the fluidics drawer and the sample injection port SIP sample Injection port SIP fluid control panel power switch Figure 2 1 FACSCalibur sensor unit Chapter 2 Getting Started 12 Power Switch The Power switch located on the bottom right side of the instrument turns the FACSCalibur instrument on and off Fluid Control Panel The fluid control panel houses the flow rate buttons and fluid control buttons used to set sample flow rate and fluid modes All instrument adjustments for the FACSCalibur are controlled through the sof
98. tton 13 22 58 64 t target cell 77 threshold secondary 59 Threshold window 33 41 Time Delay Calibration 64 69 time delay electronics 58 64 tube support arm 13 14 22 f figure u V W upgrades options and 7 vent valve toggle switch 15 waste reservoir 15 108 emptying 19 21 waste tubing 15 19 WorklistManager 7 X Y Z X parameter 38 Y parameter 38 FACSCalibur System User s Guide I8I Index 182
99. tton 37 Acquire menu 37f Acquisition Control window 37f 87 Acquisition library 6 air bubbles 21 22 air filter 143f cleaning 143 144 air supply tubing 15 16 amplifiers 32 APC beads 59 71 Apple Operating System 26 argon ion laser 11 24 55 aseptic sorting 98 102 Attractors 8 b ball valve 15 Bal seal 13 144 changing 144 146 beads APC 59 71 CaliBRITE 35 BDMAC 6 BDPAC 6 biohazardous material 19 94 biological safety x bleach 134 136 138 bovine serum albumin BSA 85 BSA bovine serum albumin 85 C CaliBRITE beads 35 catcher tube 77 78f Cell Concentrator Module 105f cleaning 127 130 components 105 108 preparing to sort 108 111 sorting with 112 FACSCalibur System User s Guide cell culture insert 108f installing holder 115f preparing 109 cell of interest 77 CELLQuest software 5 6 26 31 cleaning Concentrator Vessel 127 130 daily 133 135 monthly 135 138 periodic 139 sort line 94 97 122 collection port 106 collection station 106 collection tubes 85 Compensation 33 adjusting 48 51 72 74 Compensation window 34 41 concentrator module 105 130 Concentrator Vessel 107f cleaning 127 130 CONSORT generated data 5 27 Counters window 42 43 Cytometer menu 40 d daily cleaning 133 135 DDM parameter 63 dead sort volume 120 detectors adjusting FL1 FL2 FL3 45 48 Detectors Amps window 31 32f detectors voltages 32 dichroic mirrors 24 distilled water 22 82 DNA 4 5 25 dot plot 37 38 dual
100. tware except for the power switch and the buttons in the fluid control panel flow rate buttons fluid control buttons Figure 2 2 Fluid control panel e Flow rate buttons Three buttons LO MED HI that allow control of the sample flow rate through the flow cell 12 uL 3 uL min of sample 35 uL 5 uL min of sample and 60 uL 7 uL min of sample respectively e Fluid control buttons Three buttons RUN STNDBY PRIME that allow selection of fluidic modes RUN pressurizes the sample tube to transport the cell suspension 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 instrument switches to an automatic standby status to conserve sheath fluid the RUN button changes to orange FACSCalibur System User s Guide STNDBY standby restricts fluid flow and reduces the blue laser power to conserve sheath fluid and prolong laser life PRIME prepares the fluidics to begin a run 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 reservoir After a preset time the flow cell fills automatically with sheath fluid at a controlled rate to prevent bubble formation or entrapment At completion the instrument goes into standby mode Sample Injection Por
101. ucture and calls for no special work area safety requirements Nevertheless United States regulations require the following warning be posted to avoid tampering with the instrument DANGER LASER RADIATION WHEN OPEN AVOID DIRECT EXPOSURE TO BEAM Use of controls adjustments or performance of procedures other than those specified in this user s guide may result in hazardous laser radiation exposure Do not remove protective housing Laser power up to 15 mW at 635 nm and or 15 mW at 488 nm ina beam with a full angle divergence of 0 94 mrad could be accessible in the interior if the excitation optics cover is removed Biological Safety Blood samples may contain infectious agents that are hazardous to your health Follow appropriate biosafety procedures wear gloves when handling blood products or any materials with which they come in contact Dispose of waste reservoir contents only after it has been exposed to bleach for a minimum of 30 minutes Always follow local state and federal biohazard handling regulations when disposing of biohazardous waste material After running samples on the instrument dispose of the sample tubes in accordance with local state and federal biohazard handling regulations FACSCalibur System User s Guide Electromagnetic Compatibility Refer to European EMC Electromagnetic Compatibility Directive 89 336 EEC e This equipment conforms to EN 50082 2 EN 55011 Class A Emissions Heavy Industri
102. uide Use the table of contents and index to locate instructions for specific procedures Use the Quick Reference Guide located in the jacket pocket of this user s guide when you become familiar with the system and procedures Here s what you ll find in this user s guide e Safety and Limitations following this section contains important information youll need to know before operating the FACSCalibur system e Chapter 1 Introduction defines the FACSCalibur system giving an overview of the FACSCalibur instrument the FACStation data management system and the software that comes installed e Chapter 2 Getting Started provides you with the instructions necessary for starting up the FACSCalibur instrument and preparing it for use Also in this chapter are instructions for turning on the computer and starting the software e Chapter 3 Instrument Setup for Acquisition of Samples describes how to access instrument controls using CELLQuest software how to optimize and save instrument settings and provides instructions for setting up the FACSCalibur system to run samples and collect data for multicolor analysis e Chapter 4 FL4 Option provides instructions necessary for setting up the FACSCalibur system to run samples and collect data for 4 color analysis V1 FACSCalibur System User s Guide e Chapter 5 Sorting Option describes how to set up start and end sorting It also describes how to concentrate the sorted sa
103. ur approximately 500 mL of sterile 1X PBS into the reservoir Swirl to wash out any remaining EtOH empty the reservoir and repeat 100 16 17 18 19 20 21 FACSCalibur System User s Guide Fill the reservoir with 3 L of sterile 1X PBS Cap the reservoir before removing it from the hood Install the reservoir in the instrument Place three new collection tubes in the collection station Install a tube of sterile PBS on the SIP Click Acquire in the Acquisition Control window Run the sterile PBS for approximately 10 minutes to wash residual EtOH out of the lines Allow approximately 15 mL of PBS to run into each collection tube Achieve this by removing each tube from left to right after it fills with 15 mL of PBS 101 Chapter 5 Sorting Option 102 ae 23 24 25 Click Pause and then Abort Using aseptic technique coat the appropriate number of 50 mL conical tubes with sterile PBS 4 BSA buffer See Section 5 2 Preparing Collection Tubes for instructions Place the prepared conical tubes in the collection station Follow the steps in Section 5 6 Sorting the Sample to sort the sample CHAPTER 6 Cell Concentrator Module Option CHAPTER 6 Summary E components of the Cell Concentrator Module E preparing to sort E sorting with the Cell Concentrator Module 104 6 1 waste waste reservoir FACSCalibur System User s Guide The Cell Concentrator Module option is
104. using proper aseptic technique e 3 Lof 70 ethanol EtOH dilute in sterile distilled water e 5 Lof sterile 1X PBS 2 Fill a clean sheath reservoir with 3 L of 70 EtOH Work under a hood and use aseptic technique _ NOTE For information on removing and installing the reservoirs see Section 2 2 1 Filling the Sheath Reservoir and Section 2 2 2 Emptying the Waste Reservoir 3 Cap and shake the reservoir This ensures that the entire inner surface of the reservoir is washed with EtOH FACSCalibur System User s Guide Install the reservoir in the instrument Using a squirt bottle filled with EtOH rinse off the collection station ports Place three collection tubes in the collection station Install a tube of 70 EtOH on the SIP Set a Sort Gate Follow the instructions outlined in Section 5 3 steps 1 through 6 to create a sort gate Draw an arbitrary region in the empty display to enable sorting Choose Sort Setup from the Acquire menu The Sort Setup window appears Choose the gate drawn in step 7 from the Sort Gate pop up menu 99 Chapter 5 Sorting Option l 0 Press the RUN fluid control button l l Click Acquire in the Acquisition Control window Make sure the Setup box is checked l Run the EtOH on the FACSCalibur instrument until all three collection tubes are filled l 3 Click Pause then Abort and disconnect the reservoir l 4 Working under a hood empty the remaining EtOH l 5 Po
105. ve at the electronics simultaneously To acquire biological samples BDIS recommends that you optimize instrument settings with your samples after performing Time Delay Calibration and the FL4 setup procedures CHAPTER 5 Sorting Option CHAPTER 5 Summary sorting with the FACSCalibur system priming the sort line preparing collection tubes creating a sort gate selecting a sort gate using the Sort Counters window sorting the sample ending sorting recovering sorted cells cleaning the sort line aseptic sorting FACSCalibur System User s Guide This chapter explains how the FACSCalibur system equipped with the Sorting option sorts cells and how to choose the sort mode that fits your particular needs You can then follow the setup procedure to prepare for sorting Sorting with the FACSCalibur System When equipped with the Sorting option the FACSCalibur system uses a mechanical device called a catcher tube to sort cells This catcher tube is located in the upper portion of the flow cell and moves in and out of the sample stream to collect desired cells at a rate of up to 300 per second As a cell passes through the laser the FACSCalibur electronics system using the sort gate characteristics quickly determines whether that cell is a cell of interest target cell The target cell is then captured according to the preselected sort mode Because laser alignment and stream velocity are fixed the time it takes for desired c
106. w Click and drag each new dot plot to a clear area near the FSC vs SSC dot plot 45 Chapter 3 Instrument Setup for Acquisition of Samples l 8 Set Mode to Log for FL1 FL2 and FL3 in the Detectors Amps window Notice the axes of the plot change to a four decade logarithmic scale This allows you to cover the wide dynamic range of immunofluorescence signals You cannot adjust the amplifier gain when in Log mode l 9 Adjust the FL1 and FL2 PMT voltages Place the bead population in the lower left corner of the plot Figure 3 10 Beads 004 Figure 3 10 Adjusted FL1 FL2 voltages 2 0 Place quadrant markers on the FL1 vs FL2 dot plot Use the Quadrant Marker tool from the Tool palette to place markers as they appear in Figure 3 11 FACSCalibur System User s Guide Beads 004 Quadrant Marker tool Figure 3 11 Quadrant markers placed 2 1 Adjust the FL3 PMT voltage for the FL2 vs FL3 dot plot Place the bead population in the lower left corner of the dot plot Beads 004 Figure 3 12 Adjusted FL3 voltage 47 Chapter 3 Instrument Setup for Acquisition of Samples 2 2 Place quadrant markers on the FL2 vs FL3 dot plot gt The next step is to adjust Compensation 2 3 Install a tube of freshly mixed CaliBRITE beads on the SIP Mixed CaliBRITE beads include unlabeled FITC PE and PerCP stained beads 2 4 Adjust the FL2 FL1 compensation while viewing the FL1 vs FL2 plot Increase the FL2
107. w a region around the population you wish to sort You can continue to create regions and combine them to create a sort gate Refer to the CELL Quest Software Users Guide for details on drawing regions and creating logical gates 5 4 Selecting a Sort Gate The Sort Setup window allows you to control all sorting options by selecting the gate to be used for sorting the number of cells to be sorted and the sort mode l Choose Sort Setup from the Acquire menu The Sort Setup window appears Sort Setup Sort Gate Sort Count Aborted Cells 88 FACSCalibur System User s Guide 2 Click the Sort Gate pop up menu Choose a sort gate The subset of data in this gate will be sorted into the collection tubes If you choose No Gate you can acquire and analyze cells without sorting them 3 Enter the number of cells you want to sort in the Sort Count field A zero allows continuous sorting 4 Choose a Sort Mode from the pop up menu Select among Single Cell Recovery or Exclusion 5 Choose List or No List from the Aborted Cells pop up menu List or No list acquires to the computer the data from events that meet the abort criteria these events are identified as having physical characteristics that interfere with the detection process If you choose List data from the aborted events are saved to the computer 89 Chapter 5 Sorting Option 6 Click OK when finished 5 5 Using the Sort Counters Window Use t
108. will prevent the accumulation of saline deposits from forming in the line Disconnect the tubing from the syringe by twisting the luer end counterclockwise See Figure 5 4 2 Fill the syringe with distilled water Place the syringe nozzle in a container filled with distilled water and slowly pull up the syringe plunger until the syringe is full FACSCalibur System User s Guide Bleed any air out of the syringe by holding it luer end up and gently pushing in the plunger Reconnect the tubing to the syringe by turning it clockwise Disconnect the upper tubing of the sheath filter by squeezing the metal clip on the quick disconnect and pulling the connector from the fitting Connect the tubing from the syringe to the upper connector of the sheath filter by pushing firmly until you hear a click Figure 5 4 VENT VALVE il R PRESS TO RELIEVE PRESSURE luer end ie Figure 5 4 Syringe connected to sheath filter connector 95 Chapter 5 Sorting Option 7 Install a collection tube in the collection tube station 1 leftmost position 8 Install a tube of distilled water on the SIP 9 Press the RUN fluid control button l 0 Press the sort line purge button located inside the FACSCalibur collection station to flush the sort line See Se
109. ymphocytes Many of the unlabeled beads can still be in the first few channels when gain is properly set for FL4 You should take care when attempting to set PMT voltages on the signal from unlabeled beads or unstained cells The large number of events in very low channels can affect population means BDIS recommends you set gains using a positive population if target channels are used to judge correct setup 7A Chapter 4 FL4 Option 2 6 Remove the tube of APC beads from the SIP 2 7 Choose Compensation from the Cytometer menu The Compensation window appears gt The next step is to adjust compensation To do this proceed with step 28 or refer to the APC Beads package insert for a more quantitative method 2 8 Install a tube of freshly mixed beads on the SIP Mixed beads contain PerCP labeled CaliB RITE beads and APC beads You can make this tube by adding a drop of PerCP labeled CaliBRITE beads to the tube containing APC beads that you removed from the SIP in step 26 APC appears primarily in the FL4 detector but some of its fluorescence overlaps into the FL3 detector PerCP appears in the FL3 detector but some of its fluorescence overlaps into the FL4 detector See Figure 4 2 Use the Compensation window to adjust for this fluorescence overlap 72 FACSCalibur System User s Guide 2 9 Adjust the FL3 FL4 compensation while viewing the FL3 vs FL4 plot Adjust to rid the FL3 detector of FL4 fluorescence overlap T

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