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Becton Dickinson FACSCalibur User Manual

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Becton Dickinson
Immunocytometry Systems
2350 Qume Drive
San Jose, CA 95131-1807
Ordering Information (800) 223-8226
Customer Support Center
(800) 448-2347 (BDIS)
FAX (408) 954-2347 (BDIS)
FACSCalibur
User's Guide
02-61760-02
August, 1996
Becton Dickinson
Canada, Inc.
2464 South Sheridan Way
Mississauga, Ontario
L5J 2M8
Canada
Tel (905) 822-4820
FAX (905) 822-2644
™
Becton Dickinson
European HQ
Denderstraat 24
B-9320 Erembodegem-Aalst
Belgium
Tel (32) 53-720211
FAX (32) 53-720450
11-10823-02 Rev. A
System
Nippon Becton Dickinson
Company, Ltd.
DS Bldg
5-26, Akasaka 8-chome
Minato-ku, Tokyo 107
Japan
Tel (81) 3-5413-8251
FAX (81) 3-5413-8155
Becton Dickinson
Worldwide, Inc.
30 Tuas Avenue #2
Singapore, 2263
Tel (65) 861-0633
FAX (65) 860-1590

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  • Page 1 11-10823-02 Rev. A ™ FACSCalibur System User’s Guide 02-61760-02 August, 1996 Becton Dickinson Becton Dickinson Becton Dickinson Nippon Becton Dickinson Becton Dickinson Company, Ltd. Worldwide, Inc. Immunocytometry Systems Canada, Inc. European HQ 2350 Qume Drive 2464 South Sheridan Way Denderstraat 24...
  • Page 3 FACSCalibur User’s Guide Copyright © 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:...

  • Page 5: Table Of Contents

    Components of the Basic FACSCalibur System ....... . . 4...
  • Page 6 Sorting with the FACSCalibur System ........
  • Page 7 Appendix A Consumables and Service Information ..... . 163 Appendix B FACSCalibur Specifications ......169...

  • Page 9: Preface

    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 C Quest™...
  • Page 10 • 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.
  • Page 11 • 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. • Appendix B, FACSCalibur Specifications, provides a more detailed description of the instrument. Conventions Used in This Guide Italics Highlights any text that appears on the screen.
  • Page 12 Use the table of contents and index to locate this information. 2. See Chapter 7 for troubleshooting information. 3. US customers call the Becton Dickinson Immunocytometry Systems Customer Support Center at (800) 448-2347 (BDIS). Customers outside the US contact your local Becton Dickinson representative or distributor.

  • Page 13: 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 C Quest Software User’s Guide, and any accompanying manual for accessories and optional equipment.
  • Page 14 Safety and Limitations Laser Safety • The FACSCalibur instrument is a Class I laser product. The laser is fully contained within the instrument structure 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.
  • Page 15 FACSCalibur System User’s Guide Electromagnetic Compatibility (Refer to European EMC [Electromagnetic Compatibility] Directive 89/336/EEC) • This equipment conforms to EN 50082-2/EN 55011 Class A Emissions (Heavy Industrial Environment). It shall not be used in the residential, commercial, and light industrial environment unless the apparatus also...
  • Page 16 Safety and Limitations...

  • Page 17: Chapter 1 Introduction

    CHAPTER 1 Introduction...
  • Page 18 CHAPTER 1 Summary introduction intended use components of basic system, hardware and software installation options and upgrades...
  • Page 19 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.

  • Page 20: Intended Use

    In addition, the FACSCalibur system can be used for many research applications, including multicolor analysis, classification studies of chromosomes, DNA content analysis, platelet studies, and investigation of intracellular ionized calcium measurements.

  • Page 21: Installation

    • ModFit LT™ software, version 1.0 or later, for DNA analysis NOTE: See Appendix A, Consumables and Service Information, for a list of operating supplies necessary for using the FACSCalibur system. See Section 1.4 for application-specific software options available from BDIS.
  • Page 22 Chapter 1: Introduction When C Quest software is installed before shipment, the supporting files are placed in the appropriate folders of the computer. Performing acquisition using the Macintosh PowerPC requires the presence of the Acquisition Library (AcqLibPPC) and the BDPACDriver in the Extensions folder.

  • Page 23: Options And Upgrades

    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.
  • Page 24 Chapter 1: Introduction FACStation Software The following application-specific software programs are available from BDIS for use with the FACSCalibur system: • SimulSET™ software—for automated acquisition and analysis of two-color immunophenotyping • Attractors™ software—for innovative hierarchical data analysis automation ™ • PAINT-A-GATE software—for exploratory multidimensional data...

  • Page 25: Chapter 2 Getting Started

    CHAPTER 2 Getting Started...
  • Page 26 CHAPTER 2 Summary FACSCalibur instrument overview fluidics system components optical system components electronics system FACStation data management system overview...

  • Page 27: 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.
  • Page 28 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 software except for the power switch and the buttons in the fluid control panel.
  • Page 29 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.

  • Page 30: Fluidics Drawer Components

    Chapter 2: Getting Started • 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.
  • Page 31 FACSCalibur System User’s Guide The fluidics drawer (see 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.

  • Page 32: Filling The Sheath Reservoir

    Filling the Sheath Reservoir 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.
  • Page 33 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.
  • Page 34 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 in the Status window should return to its normal value.

  • Page 35: Emptying The Waste Reservoir

    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.
  • Page 36 Chapter 2: Getting Started Remove the waste reservoir. 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.

  • Page 37: Priming The Fluidics

    FACSCalibur System User’s Guide Install the reservoir. Snap the waste and air vent tubing into their color-coded fittings by pushing firmly until you hear a click. 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.

  • Page 38: Leaving The Facscalibur Instrument

    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.

  • Page 39: Optical System Components

    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. Optical System Components Figure 2-5 is a simplified diagram of the optical system used in the FACSCalibur. 530/30 488/10...

  • Page 40: Electronics System

    Chapter 2: Getting Started 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.

  • Page 41: Facstation Data Management System

    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.
  • Page 42 FACStation computer, refer to the appropriate software user’s guide. • Apple Operating System 7.5 software, or later • FACSComp software—instrument setup and performance evaluation program that assists in setting up the FACSCalibur instrument for immunophenotyping. • C Quest 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.
  • Page 43 If you are new to the Macintosh, refer to the Macintosh User’s 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.
  • Page 44 • 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 •...

  • Page 45: Chapter 3 Instrument Setup For Acquisition Of Samples

    CHAPTER 3 Instrument Setup for Acquisition of Samples...
  • Page 46 CHAPTER 3 Summary accessing instrument controls optimizing instrument settings saving instrument settings...

  • Page 47: Accessing Instrument Controls In Cell Quest

    C Quest 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 C Quest software.
  • Page 48 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. 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.
  • Page 49 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.
  • Page 50 Chapter 3: Instrument Setup for Acquisition of Samples FL1 (530/30) FL2 (585/42) FL3 (650) FITC PerCP 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.

  • Page 51: Optimizing The Instrument Settings

    FACSCalibur System User’s Guide 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.
  • Page 52 Chapter 3: Instrument Setup for Acquisition of Samples Choose C Quest from the Apple ( ) menu to launch the software. The C Quest desktop appears, displaying an untitled Experiment document. Menu bar Tool palette Figure 3-3 C Quest 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.
  • Page 53 FACSCalibur System User’s Guide Choose Connect to Cytometer from the Acquire menu. The Acquisition Control window appears. 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.
  • Page 54 Chapter 3: Instrument Setup for Acquisition of Samples Figure 3-4 Dot Plot dialog box 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.
  • Page 55 FACSCalibur System User’s Guide Figure 3-5 Choosing an acquisition dot plot Figure 3-6 Choosing parameters Click OK. The dot plot appears in the Experiment document.
  • Page 56 Chapter 3: Instrument Setup for Acquisition of Samples ð 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. Choose Detectors/Amps from the Cytometer menu. The Detectors/Amps window appears.
  • Page 57 FACSCalibur System User’s Guide 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.
  • Page 58 Events/Second rate may be erratic. It is important to wait for it to stabilize; it will take approximately 5 seconds. Push the RUN button on the FACSCalibur flow cytometer. Make sure the button turns green in color. If it does not, see Chapter 8,...
  • Page 59 FACSCalibur System User’s Guide 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.
  • Page 60 Chapter 3: Instrument Setup for Acquisition of Samples 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).
  • Page 61 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.
  • Page 62 Chapter 3: Instrument Setup for Acquisition of Samples 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.
  • Page 63 FACSCalibur System User’s Guide Quadrant Marker tool Figure 3-11 Quadrant markers placed 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. Figure 3-12 Adjusted FL3 voltage...
  • Page 64 Chapter 3: Instrument Setup for Acquisition of Samples Place quadrant markers on the FL2 vs FL3 dot plot. ð The next step is to adjust Compensation. Install a tube of freshly-mixed CaliBRITE beads on the SIP. Mixed CaliBRITE beads include unlabeled, FITC-, PE-, and PerCP-stained beads.
  • Page 65 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.
  • Page 66 Chapter 3: Instrument Setup for Acquisition of Samples Figure 3-14 Adjusted FL1–%FL2 compensation Figure 3-15b Adjusted compensa- Figure 3-15a Unadjusted compensation 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.

  • Page 67: Saving The Instrument Settings

    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.
  • Page 68 Chapter 3: Instrument Setup for Acquisition of Samples Click Save. A standard directory dialog box appears. 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. Click Save.

  • Page 69: Chapter 4 Fl4 Option

    CHAPTER 4 FL4 Option...
  • Page 70 CHAPTER 4 Summary FL4 optics time-delay electronics dual threshold setting up the FACSCalibur instrument for 4-color analysis time-delay calibration...

  • Page 71: Optics

    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.
  • Page 72 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.
  • Page 73 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.

  • Page 74: Time-Delay Electronics

    Chapter 4: FL4 Option Time-Delay Electronics The spatial separation of the beams results in a single particle generating signals at different moments 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.

  • Page 75: Dual Threshold

    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.

  • Page 76: Turning On The Red-Diode Lase

    Chapter 4: FL4 Option 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.
  • Page 77 FACSCalibur System User’s Guide Choose Instrument Settings from the Cytometer menu The Instrument Settings dialog box appears. 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.
  • Page 78 Chapter 4: FL4 Option Click Set. The instrument settings are sent to the FACSCalibur flow cytometer. Click Done. The Instrument Settings window disappears. ð The next step is to turn on the red diode laser. Choose Detectors/Amps from the Cytometer menu.
  • Page 79 FACSCalibur System User’s Guide 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.
  • Page 80 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.
  • Page 81 FACSCalibur System User’s Guide 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.
  • Page 82 Chapter 4: FL4 Option Figure 4-7 Time-Delay Calibration document Adjust the FSC threshold to 200 using the slider pop-up. See Figure 4-8.
  • Page 83 FACSCalibur System User’s Guide 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.
  • Page 84 Chapter 4: FL4 Option Choose Log as the Mode for FL4. Adjust the FL4 PMT voltage to place the mean peak in the FL4 histogram to Channel 800 ±5. Choose Time-Delay Calibration from the Cytometer menu. The Time-Delay Calibration dialog box appears.
  • Page 85 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. NOTE: If calibration is not successful, the dialog box disappears and an error message dialog appears.

  • Page 86: Setting Up The Fl4 Parameter

    Chapter 4: FL4 Option Setting Up the FL4 Parameter Create a FL3 vs FL4 acquisition dot plot. See Section 3.1 or refer to the C Quest Software User’s Guide for instructions on creating dot plots. Place quadrants on the FL3 vs FL4 plot. Use the Quadrant Marker tool to place markers as they appear in Figure 4-9.
  • Page 87 FACSCalibur System User’s Guide Install a tube of APC beads on the SIP. If necessary, adjust the FL4 PMT to place the bead population in the target channel recommended in the APC Beads package insert. Figure 4-10 Adjusted FL4 voltage There is little or no FL4 autofluorescence from unlabeled beads.
  • Page 88 Chapter 4: FL4 Option Remove the tube of APC beads from the SIP. Choose Compensation from the Cytometer menu. The Compensation window appears. ð 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 Install a tube of freshly mixed beads on the SIP.
  • Page 89 FACSCalibur System User’s Guide Adjust the FL3–%FL4 compensation while viewing the FL3 vs FL4 plot. Adjust to rid the FL3 detector of FL4 fluorescence overlap. To 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).
  • Page 90 Chapter 4: FL4 Option 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 arrive at the electronics simultaneously.

  • Page 91: Chapter 5 Sorting Option

    CHAPTER 5 Sorting Option...
  • Page 92 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...

  • Page 93: Sorting With The Facscalibur System

    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).

  • Page 94: Choosing A Sort Mode

    Chapter 5: Sorting Option catcher tube 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.
  • Page 95 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.
  • Page 96 Chapter 5: Sorting Option 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.
  • Page 97 FACSCalibur System User’s Guide Target cell capture above 300 cells/sec not possible 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...

  • Page 98: Priming The Sort Line

    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.
  • Page 99 Install a 50-mL tube in the first collection port on the left. first collection port sort line purge button 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.
  • Page 100 Chapter 5: Sorting Option Remove the 50-mL tube from the first collection port and place it in the middle collection port. Repeat step 3. Remove the 50-mL tube from the middle collection port and place it in the third collection port on the right. Repeat step 3.

  • Page 101: Preparing Collection Tubes

    FACSCalibur System User’s Guide 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. Fill one to three 50-mL conical tubes with a 4% BSA solution.

  • Page 102: Creating A Sort Gate

    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 Creating a Sort Gate Gates defined in C Quest software can be used for acquisition, analysis, and sorting.
  • Page 103 FACSCalibur System User’s Guide Choose Connect to Cytometer from the Acquire menu. The Acquisition Control window appears. The Setup box should be checked. Install the sample tube on the SIP, quickly center the tube support arm under the tube, and press the RUN fluid control button.

  • Page 104: Selecting A Sort Gate

    Chapter 5: Sorting Option Click in the plot and draw 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 C Quest Software User’s Guide for details on drawing regions and creating logical gates.
  • Page 105 FACSCalibur System User’s Guide 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.

  • Page 106: Using The Sort Counters Window

    Chapter 5: Sorting Option Click OK when finished. Using the Sort Counters Window Use the 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. Choose Sort Counters from the Cytometer menu.

  • Page 107: Sorting The Sample

    FACSCalibur System User’s Guide • Auxiliary Rate/Auxiliary Total—displays the rate (events/sec) or the total number of cells the FACSCalibur system is processing; includes the aborted cells. • Sort Rate/Sort Total—displays the rate (events/sec) or the total number of cells that are sorted.

  • Page 108: Ending Sorting

    Chapter 5: Sorting Option Click Acquire in the Acquisition Control window. As the sample is sorted, a static-like sound indicates sorting is taking place. Sorting stops when the first of four conditions is met. See Section 5.7, Ending Sorting, for details. Ending Sorting There are four ways to end sorting.

  • Page 109: Recovering Sorted Cells

    FACSCalibur 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.

  • Page 110: Cleaning The Sort Line

    Chapter 5: Sorting Option Resuspend the pellet with 100 µL 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.
  • Page 111 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.
  • Page 112 Install a tube of distilled water on the SIP. Press the RUN fluid control button. Press the sort line purge button, located inside the FACSCalibur collection station, to flush the sort line. See Section 5.1, Priming the Sort Line, for more details.
  • Page 113 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.

  • Page 114: 5.10 Aseptic Sorting

    Chapter 5: Sorting Option 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.
  • Page 115 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.
  • Page 116 Press the RUN fluid control button. Click Acquire in the Acquisition Control window. Make sure the Setup box is checked. Run the EtOH on the FACSCalibur instrument until all three collection tubes are filled. Click Pause, then Abort, and disconnect the reservoir.
  • Page 117 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.
  • Page 118 Chapter 5: Sorting Option 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.

  • Page 119: Chapter 6 Cell Concentrator Module Option

    CHAPTER 6 Cell Concentrator Module Option...
  • Page 120 CHAPTER 6 Summary components of the Cell Concentrator Module preparing to sort sorting with the Cell Concentrator Module...

  • Page 121: Cell Concentrator Module Components

    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.
  • Page 122 Chapter 6: Cell Concentrator Module Option 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. air pressure adjustment knob power button air pressure button...
  • Page 123 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.

  • Page 124: Preparing The Cell Concentrator Module To Sort

    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.
  • Page 125 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 pressure. Cell culture inserts should be coated with a bovine serum albumin (BSA) solution before use.
  • Page 126 Chapter 6: Cell Concentrator Module Option Cover the tissue culture plate, and store overnight in the refrigerator. ° Inserts may be stored at 4 C for up to 1 month. Just before using the insert, rinse it with 1X PBS Pour out the BSA solution.
  • Page 127 FACSCalibur System User’s Guide Place the white O-ring into the top of the holder. 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.

  • Page 128: Sorting With The Cell Concentrator Module

    Chapter 6: Cell Concentrator Module Option 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.

  • Page 129: Determining Reference Pressure

    FACSCalibur System User’s Guide Install a tube of PBS on the SIP, and set the fluid control to RUN. Press the sort line purge button. See Section 5.1, Priming the Sort Line. If there is no clog in the sort line, you should see fluid dripping from the sort line into the lower chamber of the concentrator vessel.
  • Page 130 Chapter 6: Cell Concentrator Module Option 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. Place a rubber O-ring into the retainer in the lower compartment of the vessel.
  • Page 131 FACSCalibur System User’s Guide O-ring cell culture insert holder 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.
  • Page 132 Chapter 6: Cell Concentrator Module Option 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.
  • Page 133 FACSCalibur System User’s Guide Adjust the pressure by turning the air pressure adjustment knob (Figure 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.

  • Page 134: Sorting And Concentrating Cells

    Chapter 6: Cell Concentrator Module Option Sorting and Concentrating Cells Add 100 mL of undiluted bleach to the empty waste reservoir before sorting into the concentrator vessel. Make sure the Cell Concentrator Module is on and the concentrator vessel is properly assembled with the cell culture insert properly installed. Make sure the LO flow rate is selected.
  • Page 135 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. Adjust the pressure to the predetermined reference value by turning the air pressure adjustment knob (Figure 6-2).

  • Page 136: Recovering Sorted Cells From The Sort Line

    Chapter 6: Cell Concentrator Module Option 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. Remove cells from the insert for further processing or reanalysis. BDIS has not optimized, and therefore does not support, techniques for using the Cell Concentrator Module to recover viable cells.
  • Page 137 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.

  • Page 138: Removing Cells For Re-Analysis

    Chapter 6: Cell Concentrator Module Option Removing Cells for Re-analysis Open the concentrator vessel. Unscrew the top of the vessel by turning counterclockwise. 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.
  • Page 139 FACSCalibur System User’s Guide 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. Disconnect the tubing from the syringe by twisting the luer end counterclockwise.
  • Page 140 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.
  • Page 141 FACSCalibur System User’s Guide Place a tube of distilled water on the SIP. Set the fluid control to RUN. Press the sort-line purge button. 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.
  • Page 142 Chapter 6: Cell Concentrator Module Option Remove the collection tube. Turn on the Cell Concentrator Module by pressing the power button. Repeat 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.

  • Page 143: Cleaning The Concentrator Vessel

    FACSCalibur System User’s Guide Press the air pressure button to turn if off. Press the power button to turn off the Cell Concentrator Module. Drain and fill (PRIME) the flow cell two to three times to remove any air that may have entered the flow cell.
  • Page 144 Chapter 6: Cell Concentrator Module Option Disconnect the concentrator vessel 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.
  • Page 145 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.
  • Page 146 Chapter 6: Cell Concentrator Module Option 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.

  • Page 147: Chapter 7 Cleaning And Maintenance

    CHAPTER 7 Cleaning and Maintenance...
  • Page 148 CHAPTER 7 Summary daily FACSCalibur instrument cleaning monthly cleaning changing the sheath filter cleaning the air filter changing the Bal seal changing the sample O-ring...

  • Page 149: Daily Cleaning

    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.
  • Page 150 Chapter 7: Cleaning and Maintenance outer sleeve sample injection tube tube support arm Figure 7-1 Sample injection port (SIP) Move the tube support arm to the side. 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.

  • Page 151: Monthly Cleaning

    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. Center the tube support arm, and allow the water to run for 5 minutes.
  • Page 152 Chapter 7: Cleaning and Maintenance 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.
  • Page 153 FACSCalibur System User’s Guide VENT VALVE PRESS TO RELIEVE PRESSURE Saline Filter port SALINE FILTER sheath tubing Figure 7-2 Bypassing sheath filter 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.
  • Page 154 Chapter 7: Cleaning and Maintenance Remove the tube of bleach from the SIP. Repeat steps 3 through 6 using distilled water instead of the 1:10 dilution of bleach. Replace the original sheath reservoir. Reconnect the sheath filter by pushing the connector into its fitting until you hear a click.

  • Page 155: Periodic Maintenance

    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.
  • Page 156 Chapter 7: Cleaning and Maintenance output quick-disconnect vent port O-ring filter base input quick-disconnect Figure 7-3 Sheath filter 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.
  • Page 157 FACSCalibur System User’s Guide Unscrew the base of the filter to remove 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.
  • Page 158 Chapter 7: Cleaning and Maintenance 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.

  • Page 159: Cleaning The Air 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.

  • Page 160: Changing The Bal Seal

    Chapter 7: Cleaning and Maintenance 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.
  • Page 161 FACSCalibur System User’s Guide Bal seal Figure 7-4 Removing outer sleeve Figure 7-6 Removing Bal seal Install the new Bal seal spring-side up.
  • Page 162 Chapter 7: Cleaning and Maintenance 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.

  • Page 163: Changing The Sample O-Ring

    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.
  • Page 164 Chapter 7: Cleaning and Maintenance 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. Install a sample tube on the SIP.

  • Page 165: Chapter 8 Troubleshooting

    CHAPTER 8 Troubleshooting...
  • Page 166 CHAPTER 8 Summary symptoms you might observe during instrument operation; possible causes, and solutions...
  • Page 167 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...
  • Page 168 Replace Bal seal. See Section 7.2.5. Cause: Communication failure between computer and FACSCalibur instrument. Solution: Turn off computer and FACSCalibur instrument. Turn on instrument, followed by computer. Cause: GPIO error, cannot read instrument status. Solution: Turn off computer and FACSCalibur instrument. Reseat GPIO cable, located next to power cord in back of cytometer.
  • Page 169 Laser not functioning. Solution: 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. Cause: Leak at sheath area.
  • Page 170 Chapter 8: Troubleshooting Cause: Sheath reservoir empty or waste reservoir full. Solution: Check reservoirs, fill sheath and empty waste, if necessary. See Section 2.2.1 and Section 2.2.2. Cause: Electronic connector for the sheath or waste reservoir is not properly seated in the connector port. Solution: Firmly seat the connector into its port.
  • Page 171 FACSCalibur System User’s Guide Low sample event rate YMPTOM Cause: Threshold level too high. Solution: Lower threshold level. Cause: Threshold parameter gain setting too low. Solution: Increase threshold parameter gain setting. Cause: Sample not adequately mixed. Solution: Mix sample to suspend cells.
  • Page 172 Chapter 8: Troubleshooting Cause: Contaminated sample. Solution: Prep specimen again, making sure tube is clean. Scatter parameters appear distorted YMPTOM Cause: Instrument settings adjustment is necessary. Solution: Perform optimization procedure. Cause: Air bubble in flow cell. Solution: Press PRIME to drain and fill the flow cell. See Section 2.2.3, step 2. Cause: Air in sheath filter.
  • Page 173 FACSCalibur System User’s Guide Cause: Sheath filter dirty. Solution: Change sheath filter. See Section 7.2.3. Cause: Flow cell dirty. Solution: Perform monthly maintenance (see Section 7.1.2), or wash with 10% bleach and warm deionized water. Cause: Sample contains excessive amount of debris.
  • Page 174 Chapter 8: Troubleshooting Cells not viable after sorting YMPTOM 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.
  • Page 175 FACSCalibur System User’s Guide Cause: Air bubbles in sample when reanalyzed. Solution: Gently mix the sample to avoid air bubble formation before reanalyzing. Cause: Air bubbles in flow cell. Solution: Press PRIME to drain and fill flow cell. See Section 2.2.3, step 2.
  • Page 176 Chapter 8: Troubleshooting Cause: Sample preparation technique questionable. Solution: Seek advice on sample preparation techniques. Cause: Air bubbles in sheath filter. Solution: Push the roller in the pinchcock forward to purge air from the filter. Cause: Sample not diluted in same fluid as sheath fluid. Solution: Dilute sample in the same fluid as you are using for sheath.
  • Page 177 FACSCalibur System User’s Guide Flow cell will not fill YMPTOM Cause: No sheath pressure. Solution: 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.
  • Page 178 Chapter 8: Troubleshooting Cause: Air in sheath filter. Solution: Vent air from sheath filter. See Section 2.2.3, step 2. Cause: Air bubble in flow cell. Solution: Press PRIME to drain and fill the flow cell. See Section 2.2.3, step 2. Cause: Timing between blue and red laser is inaccurate.

  • Page 179: Appendix A Consumables And Service Information

    π Appendix A Consumables and Service Information...
  • Page 180 Appendix A: Consumables and Service Information...
  • Page 181 FACSCalibur System User’s Guide A.1 Supplies Required Flow Cytometry Applications Product Description Purpose Part Number ® 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 sheath fluid...
  • Page 182 Appendix A: Consumables and Service Information For the Sorting Option Product Purpose Part Number Centrifuge with swinging-bucket rotor for concentrating cells after sorting for 50-mL conical tubes Deionized water for rinsing the sort line Phosphate-buffered saline (PBS) (Dul- for sorting applications becco’s) Bovine serum albumin (BSA) for coating collection tubes...
  • Page 183 FACSCalibur System User’s Guide For the Cell Concentrator Module Option Product Part Number Hydrophobic filter for waste reservoir 41-10015-00 Silicon tubing (sort line) 80-30012-00 Concentrator vessel sort line needle 06-30298-00 Spare waste reservoir (BDIS) 05-10941-00 12-mm insert holder 06-30299-00 25-mm insert holder...
  • Page 184 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...

  • Page 185: Appendix B Facscalibur Specifications

    Appendix B FACSCalibur Specifications...
  • Page 186 Appendix B: FACSCalibur Specifications...
  • Page 187 FACSCalibur System User’s Guide CYTOMETER SPECIFICATIONS Performance Fluorescence Sensitivity Estimated detection limit is 750 molecules of equivalent soluble fluorescein Fluorescence Resolution Coefficient of variation in FL2-Area of <3%, full peak for propidium iodide-stained chicken erythrocyte nuclei Forward and Side Scatter Sensitivity Sensitivity enables the separation of fixed platelets from noise...
  • Page 188 Appendix B: FACSCalibur Specifications Fluidics General Operation 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. Fluid Reservoirs Easily accessible 4-L capacity sheath and waste containers are housed in a convenient pull-out drawer;...
  • Page 189 FACSCalibur System User’s Guide Data Entry Sample information, reagent panels, and rack information can be defined for up to 640 tubes (40 tubes x 16 racks) at a time Loader Control Automated control through WorklistManager software and manual control with stat interrupt capability through FACS Loader electronic keypad...
  • Page 190 Appendix B: FACSCalibur Specifications REMOTE DIAGNOSTICS Remote diagnostics modem provided for direct customer support instrument interaction INSTALLATION REQUIREMENTS Power 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 ±...

  • Page 191: Index

    Index...
  • Page 192 Index...
  • Page 193 FACSCalibur System User’s Guide cell culture insert 108f installing holder 115f acquire button 37 preparing 109 Acquire menu 37f cell of interest 77 Acquisition Control window 37f, 87 Quest software 5, 6, 26, 31 Acquisition library 6 cleaning air bubbles 21, 22 Concentrator Vessel 127–130...
  • Page 194 (FSC) 24, 43 FACSConvert 5, 27 four-color acquisition 55 FACS Loader 7 four-color analysis 59–74 FACStation Data Management System 4, FACSCalibur instrument setup 59–63 25f–27 FSC (forward light scatter) 24, 43 converting files 5, 27 Experiment documents 28 g, h, i...
  • Page 195 FACSCalibur System User’s Guide saving 51–52 options threshold 59 Cell Concentrator Module 105f instrument setup 35–51 FACS Loader 7 FL4 55, 56 j, k, l Sorting 118–120 O-ring 114, 115, 141 laser overlap, spectral 33, 34f, 57f red diode 7, 56, 62...
  • Page 196 Index saline filter port 124f sorting 118–120 sample injection port (SIP) 13f, 134f aseptic 98–102 sample injection tube 14 concentrating cells 119 Bal seal 144 ending 92, 93 tube support arm 13, 14, 22 option 7, 77 Sample O-ring, changing 147, 148 preparation 82 secondary threshold 33 recovering sorted cells 93–94, 120–122...
  • Page 197 FACSCalibur System User’s Guide 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 f = figure...
  • Page 198 Index...

Table of Contents