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BD LSR II User Manual

Flow cytometer
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bdbiosciences.com
Part No. 640752 Rev. A
May 2006
BD Biosciences
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BD LSR II
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  • Page 1 BD LSR II User’s Guide bdbiosciences.com Part No. 640752 Rev. A May 2006 BD Biosciences Asia Pacific Brazil Canada 2350 Qume Drive Tel (65) 6-861-0633 Tel (55) 11-5185-9995 Tel (888) 259-0187 San Jose, CA 95131-1807 Fax (65) 6-860-1590 Fax (55) 11-5185-9895...
  • Page 2 All other company and product names might be trademarks of the respective companies with which they are associated. Patents BD LSR II is covered by one or more of the following US patents and foreign equivalents: 4,745,285; 4,844,610; and 6,014,904. PE and APC: US 4,520,110; 4,859,582; 5,055,556; Europe 76,695; Canada 1,179,942 PerCP: US 4,876,190 Cy5.5 and Cy7: US 5,268,486;...
  • Page 3 Change Made 334717 Rev. A 12/02 Initial release 338639 Rev. A 10/04 Updated software terminology and screen shots for BD FACSDiva software version 4.1 640752 Rev. A 5/06 Updated software terminology and screen shots for BD FACSDiva software version 5.0...

  • Page 5: Table Of Contents

    BD LSR II Documentation ........
  • Page 6 BD LSR II Workstation ........
  • Page 7 Chapter 3: Running Samples Sample Optimization Using Instrument Setup ......Verifying Instrument Configuration and User Preferences ... Creating an Experiment .
  • Page 8 Compensation Theory ........viii BD LSR II User’s Guide...
  • Page 9 Digital Electronics ......... BD FACSDiva Option White Paper ......
  • Page 10 4-Color Red Octagon Default Configuration Map ....BD FACS Instrument Configuration Application .....

  • Page 11: About This Guide

    About This Guide The BD LSR II User’s Guide describes the procedures necessary to operate and maintain your BD™ LSR II flow cytometer. Because many instrument functions are controlled by BD FACSDiva™ software, this guide also contains information about software features required for basic instrument setup and operation.

  • Page 12: Bd Lsr Ii Documentation

    The online help installed with your BD FACSDiva software contains the same text as that in the documents listed below, enhanced with features like full text search and related topic links. Access BD LSR II online help from the Help menu of BD FACSDiva software.

  • Page 13: Printed Documentation

    Printed Documentation A printed copy of the following documents is distributed with the BD LSR II flow cytometer: • BD LSR II User’s Guide describes procedures necessary to operate and maintain your BD LSR II flow cytometer. Because many instrument...

  • Page 14: Electronic Documentation

    Electronic Documentation PDF versions of the following documents can be found on the BD FACSDiva software installation disk or on your computer hard drive: • The BD FACSDiva Software Reference Manual includes instructions or descriptions for installation and setup, workspace components, acquisition controls, analysis tools, and data management.

  • Page 15: Technical Assistance

    • details of recent system performance For instrument support from within the US, call (877) 232-8995. For support from within Canada, call (888) 259-0187. Customers outside the US and Canada, contact your local BD representative or distributor. About This Guide...
  • Page 16 BD LSR II User’s Guide...

  • Page 17: Safety And Limitations

    The BD LSR II flow cytometer and its accessories are equipped with safety features for your protection. Operate only as directed in the BD LSR II User’s Guide and the BD LSR II Safety and Limitations booklet. Do not perform instrument maintenance or service except as specifically stated.

  • Page 18: Laser Product Classification

    The higher the classification number, the greater the potential hazard. The BD LSR II flow cytometer is a Class I (1) laser product per 21 CFR Subchapter J and IEC/EN 60825-1:1994 + A1:2003 + A2:2001. The lasers and the laser energy are fully contained within the instrument structure and call for no special work area safety requirements except during service procedures.

  • Page 19: Electrical Safety

    • For installation outside the US, use a power transformer or conditioner to convert the local power source to meet the BD LSR II power requirements (120 V ±10%, 50/60 Hz). Contact your local BD office for further information.

  • Page 20: Biological Safety

    Approved Guideline. Wayne, PA: National Committee for Clinical Laboratory Standards, 1997. NCCLS document M29-A. • Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Wayne, PA: National Committee for Clinical Laboratory Standards; 1990. NCCLS document H18-A. BD LSR II User’s Guide...

  • Page 21: General Safety

    To prevent injury by moving parts, keep your hands and clothing away from the instrument during operation. Symbols and Labels The following symbols, warnings, or precaution labels appear on the BD LSR II flow cytometer or the waste and fluid tanks. Symbol Meaning Location(s)
  • Page 22 Visible and/or where the laser beam can invisible laser emerge from the radiation when instrument removed. Avoid eye or skin exposure to direct or scattered radiation. ™ Sheath Near BD FACSFlow solution (sheath) port xxii BD LSR II User’s Guide...

  • Page 23: Limitations

    For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD Biosciences delivers software and workstations that are intended for running the instruments supplied by BD Biosciences. It is the responsibility of the buyer/ user to ensure that all added electronic files including software and transport media are virus free.
  • Page 24 BD LSR II User’s Guide...

  • Page 25: Chapter 1: Introduction

    Introduction The following topics are covered in this chapter: • Overview on page 26 • Fluidics on page 28 • Optics on page 32 • BD LSR II Workstation on page 36...

  • Page 26: Overview

    Power Switch The power switch is located on the lower-right side of the BD LSR II instrument as shown in Figure 1-1. BD LSR II User’s Guide...

  • Page 27: Control Panel

    Handles The instrument handles (Figure 1-1 on page 26) are for BD Biosciences authorized personnel only. Do not access them or attempt to lift the instrument with them, or you could injure yourself. Chapter 1: Introduction...

  • Page 28: Fluidics

    STNDBY (standby) stops fluid flow to conserve sheath fluid. When you leave the instrument for more than a few minutes, place a tube containing 1 mL of deionized (DI) water on the sample injection port (SIP) and press STNDBY. BD LSR II User’s Guide...

  • Page 29: Sample Injection Port

    • PRIME prepares the fluidics system by draining and filling the flow cell with sheath fluid. The fluid flow initially stops and pressure is reversed to force fluid out of the flow cell and into the waste container. After a preset time, the flow cell fills with sheath fluid at a controlled rate to prevent bubble formation or entrapment.
  • Page 30 If a sample tube is left on the SIP with the tube support arm to the side (vacuum on), sample will be aspirated into the waste container. BD LSR II User’s Guide...

  • Page 31: Sheath And Waste Containers

    Sheath and Waste Containers The sheath and waste containers are outside the instrument and can be positioned on the floor. Sheath Container The sheath container has a capacity of 8 L. Sheath fluid is filtered through an in- line, interchangeable filter that prevents small particles from entering the sheath fluid lines.

  • Page 32: Optics

    Optics Figure 1-4 shows the optical bench components of the BD LSR II instrument. Figure 1-4 Optical bench components (engineering model) photomultiplier tube (PMT) red 633-nm laser (optional) trigon blue 488-nm laser (standard) octagon UV 355-nm laser (optional) violet 405-nm laser...

  • Page 33: Lasers

    Lasers The BD LSR II flow cytometer has a fixed-alignment 488-nm laser with the option of additional fixed-alignment lasers. Table 1-1 BD LSR II flow cytometer laser options Warm-Up Wavelength Power Laser Type Time (Color) (mW) (min) Standard Coherent Sapphire™...

  • Page 34: Filters

    Filters Optical filters attenuate light or help direct it to the appropriate detectors. The BD LSR II instrument uses dichroic filters. Dichroic filters transmit light of a specific wavelength, while reflecting other wavelengths. The name and spectral characteristics of each filter appear on its holder.

  • Page 35: Detectors

    Figure 1-5 Dichroic filter types in octagon array bandpass filters longpass dichroic mirrors The steering optics and filters mounted on the BD LSR II instrument are listed in Table E-1 on page 154. See Optical Filters on page 127 for a more detailed explanation of how filters work in the BD LSR II flow cytometer.

  • Page 36: Bd Lsr Ii Workstation

    BD LSR II Workstation Acquisition, analysis, and most BD LSR II instrument functions are controlled by the BD LSR II workstation. It includes a PC, one or two monitors, and a printer. Your workstation is equipped with the following: •...

  • Page 37: Chapter 2: Instrument Setup

    Instrument Setup • Starting the Cytometer and Computer on page 38 • Setting Up the Optical Filters and Mirrors on page 39 • Preparing Sheath and Waste Containers on page 44 • Preparing the Fluidics on page 47 • Quality Control on page 49...

  • Page 38: Starting The Cytometer And Computer

    Allow 60 minutes for lasers to warm up and stabilize. Failure to warm up and stabilize the lasers could affect sample data. Start up the BD LSR II workstation and log in to Windows. You can turn on the power to the flow cytometer and the workstation in any order.

  • Page 39: Setting Up The Optical Filters And Mirrors

    Setting Up the Optical Filters and Mirrors Before you run samples, set up the optical filters. The following figure shows the location of the detector arrays (beneath the instrument covers of the BD LSR II flow cytometer). Each detector array is labeled with its laser source.

  • Page 40: Filter And Mirror Configurations

    (mirror slot) (filter slot) To ensure data integrity, do not leave any slots unfilled in a detector array when you are using the associated laser. Always use a blank optical holder. BD LSR II User’s Guide...
  • Page 41 Controls chapter of the BD FACSDiva Software Reference Manual for details.) BD LSR II Configuration Upgrade If you upgrade your BD LSR II cytometer with additional lasers or special order detector arrays, you will need to run the ICA software to update the BD FACSDiva database for your instrument.

  • Page 42: Changing Optical Filters Or Mirrors

    Close the instrument cover(s). Custom Configurations See Custom Configurations in Appendix E for specifications of some common custom filter and mirror configurations. The custom filters and mirrors used in these configurations are included with your spares kit. BD LSR II User’s Guide...

  • Page 43: Filter And Mirror Specifications

    Filter and Mirror Specifications Table 2-1 Longpass dichroic mirrors in octagon or trigon Specification Measurement diameter 0.625 in. +0.000, –0.005 minimum clear aperture 0.562 in. incident angle 11.25° thickness 0.125 in ±0.005 in. Table 2-2 Bandpass filters in octagon or trigon Specification Measurement diameter...

  • Page 44: Preparing Sheath And Waste Containers

    This ensures that you will not run out of sheath fluid during an experiment and that the waste container will not become too full. Preparing the Sheath Container Figure 2-3 Sheath container clamp knob air line vent valve fill point sheath tank cytometer fluid line filter assembly BD LSR II User’s Guide...
  • Page 45 Depressurize the sheath container by pulling up on its vent valve. Remove the sheath container lid. Unscrew the clamp knob and lift. Add 6 L of sheath fluid, such as BD FACSFlow™ solution, to the sheath container. NOTICE Do not fill the sheath tank to its maximum capacity (8 L). When a full tank is pressurized, erratic instrument performance can result.

  • Page 46: Preparing The Waste Container

    Empty the waste container. The waste container contents might be biohazardous. Treat contents with bleach (10% of total volume). Dispose of waste using proper precautions and in accordance with local regulations. Wear suitable protective clothing, eyewear, and gloves. BD LSR II User’s Guide...

  • Page 47: Preparing The Fluidics

    Add approximately 1 L of bleach to the waste container and close it. Reconnect the orange waste tubing and make sure it is not kinked. Reconnect the level sensor line. Preparing the Fluidics Next, make sure the fluidics system is ready. This section covers the following preparations: •...
  • Page 48 Replace the vent cap. Check the fluid lines for air bubbles. Open the roller clamp on the blue cytometer fluid line (if necessary) to bleed off any air in the line. Close the roller clamp. BD LSR II User’s Guide...

  • Page 49: Priming The Fluidics

    Priming the Fluidics Sometimes, air bubbles and debris are stuck in the flow cell. This is indicated by excessive noise in the forward scatter parameter. In these cases, it is necessary to prime the fluidics system. Remove the tube from the SIP. Press the PRIME fluid control button to force the fluid out of the flow cell and into the waste container.
  • Page 50 BD LSR II User’s Guide...

  • Page 51: Chapter 3: Running Samples

    Running Samples This chapter describes procedures that use BD FACSDiva software to record and analyze sample data: • Sample Optimization Using Instrument Setup on page 52 • Recording and Analyzing Data on page 65...

  • Page 52: Sample Optimization Using Instrument Setup

    Before attempting to perform the procedures in this chapter, you should be familiar with: • BD LSR II instrument startup, setup, and QC procedures (see Chapter 2, Instrument Setup on page 37) • BD FACSDiva software concepts: workspace components, instrument and acquisition controls, tools for data analysis.
  • Page 53 Sample optimization consists of several main steps, to be performed in the order listed below. Each of the steps is explained in greater detail in the sections that follow. Verify instrument configuration and user preferences. Create an experiment. Adjust voltages and the threshold setting. Record the compensation tubes.

  • Page 54: Verifying Instrument Configuration And User Preferences

    Preferences To obtain accurate data results, the current BD FACSDiva software instrument configuration must reflect your BD LSR II instrument optics. Verify the configuration and preferences before you create an experiment. Choose Instrument > Instrument Configuration and verify the current configuration.
  • Page 55 Figure 3-2 User Preferences dialog • Under the Templates tab, verify that the Default global worksheet checkbox is enabled. Refer to the BD FACSDiva Software Reference Manual for more information about instrument configuration and user preferences. Chapter 3: Running Samples...

  • Page 56: Creating An Experiment

    Figure 3-3 Use global instrument settings checkbox Specify the parameters for instrument setup. • In the Browser, select the Instr Settings of MyExperiment. • Make sure the parameters you need appear on the Parameters tab in the Inspector. BD LSR II User’s Guide...
  • Page 57 If more than one parameter is available for a particular PMT, you might have to select the one you need from a menu. For this example, choose PerCP from the PerCP-Cy5-5 menu. • Delete any unnecessary parameters. To delete a parameter, select it (by clicking the selection button to the left of the parameter name), and then click the Delete button.
  • Page 58 5 above. For this bead example, you do not need to provide non-generic tube labels. • Click OK to create the control tubes. Figure 3-4 Create Compensation Controls dialog BD LSR II User’s Guide...

  • Page 59: Adjusting The Voltages And Threshold

    Compensation controls are added to the experiment, along with a single stained control tube for each parameter in the experiment, and an unstained control tube (Figure 3-5). Worksheets containing appropriate plots and gates are added for each compensation tube. Figure 3-5 Compensation tubes Adjusting the Voltages and Threshold In this section, you use the unstained control tube to adjust FSC and SSC voltages and FSC threshold, to gate the population of interest (bead singlets, in this case),...
  • Page 60 To quickly adjust the gate, right-click the gate boundary, and choose Recalculate. Figure 3-6 P1 gate adjusted to singlet population Right-click the gate and choose Apply to All Compensation Controls. The P1 gate on each Stained Control worksheet is updated with your changes. BD LSR II User’s Guide...
  • Page 61 For each fluorescence parameter, adjust PMT voltages to place the negative population within the first log decade (Figure 3-8 on page 62). To adjust voltage settings, use controls in the Parameters tab in the Instrument window. Refer to the BD FACSDiva Software Reference Manual for assistance. Chapter 3: Running Samples...
  • Page 62 In order to calculate compensation, all controls must be recorded with the same PMT voltage settings. If you need to adjust the PMT voltage for a subsequent compensation control, you will need to record all compensation controls again. BD LSR II User’s Guide...

  • Page 63: Calculating Compensation

    Calculating Compensation Before you can calculate compensation, you need to record data for each single- stained control. Install the first stained control tube onto the cytometer. In the Acquisition Dashboard, click Next Tube, and then Acquire Data. Alternatively, move the current tube pointer to the next tube and click the pointer to start acquisition.
  • Page 64 MyExperiment are performed with the new compensation settings. NOTICE BD Biosciences recommends that you always visually and statistically inspect automatically calculated overlap values. The means of the positive controls should be aligned with the means of the negative.

  • Page 65: Recording And Analyzing Data

    Recording and Analyzing Data This section outlines some basic acquisition and analysis tasks using BD FACSDiva software. The example shows data from two 4-color bead samples with the following fluorochromes: • FITC • • PerCP • The procedure builds on the results obtained in the previous exercise: Sample Optimization Using Instrument Setup on page 52.
  • Page 66 When you record immunophenotyping data, use the Labels tab of the Experiment Layout dialog to provide reagent antibody labels. • Click the Acquisition tab. Verify the number of events to acquire is set to 10,000 for both tubes. • Click OK. BD LSR II User’s Guide...

  • Page 67: Recording Data

    On the MyData worksheet, create some plots for previewing data: • FSC vs SSC • FITC vs PE • FITC vs PerCP • FITC vs APC Double-click the Dot Plot button to keep the button selected until you create all plots. Recording Data In this section, you preview and record data for multiple samples.
  • Page 68 Move the current tube pointer to Beads_002. Click Acquire Data to begin acquisition. Before recording, preview the data on the MyData worksheet. Click Record Data. When event recording has completed, remove the second tube from the cytometer. BD LSR II User’s Guide...

  • Page 69: Analyzing Data

    If you are recording more than two tubes, repeat steps 7 through 11 for the additional tubes. Print the experiment-level instrument settings. Right-click the Instr Settings icon and choose Print. Analyzing Data In this section, you analyze the recorded tubes by creating plots, gates, a population hierarchy, and statistics views on a new global worksheet.
  • Page 70 Draw a gate around the PE-positive population, and name the population PE positive. • Draw a gate around the PerCP-positive population, and name the population PerCP positive. • Draw a gate around the APC-positive population, and name the population APC positive. BD LSR II User’s Guide...
  • Page 71 Format the Statistics view. • Right-click the Statistics view and choose Edit Statistics View. • Click the Header tab, and select the Specimen Name and Tube Name checkboxes. • Click the Populations tab, and select all populations except All Events; deselect the %Parent, %Total, and # Events checkboxes.
  • Page 72 Figure 3-11 Bead analysis BD LSR II User’s Guide...

  • Page 73: Reusing The Analysis

    Reusing the Analysis Global worksheets allow you to apply the same analysis to a series of recorded tubes. Once you define an analysis for a tube, use it to analyze the remaining tubes in the experiment. After viewing the data, print the analysis or save it to a tube-specific worksheet (see Saving the Analysis).
  • Page 74 Beads_001_Analysis normal worksheet. View the analysis by double- clicking the Beads_001 tube icon in the Browser. Apply the global worksheet analysis to multiple tubes (on a single normal worksheet) by selecting more than one tube before you paste the analysis. BD LSR II User’s Guide...

  • Page 75: Chapter 4: Dna Analysis

    DNA Analysis The following topics are covered in this chapter: • Criteria for DNA Experiments on page 76 • CEN Optimization on page 78 • CTN Resolution on page 84 • Optimization for Data Recording on page 86...

  • Page 76: Criteria For Dna Experiments

    On an instrument with good linearity, the doublet peak should be located at twice the mean channel of the singlet peak (Figure 4-1). Figure 4-1 Area signal and DNA fluorescence BD LSR II User’s Guide...

  • Page 77: Dna Setup

    DNA Setup In this chapter, you use the BD™ DNA QC Particles kit to verify critical DNA analysis criteria, and optimize your cytometer for DNA experiments. The instructions given here assume that DAPI is being used as described in How to Use DAPI with DNA QC, below.

  • Page 78: How To Use Pi With Dna Qc

    How to Use PI with DNA QC You can use the PI that comes with the BD DNA QC Particles kit instead of DAPI. To do so, prepare the CEN and CTN samples as described in the kit instructions. CEN Optimization Use the following procedure to set up BD FACSDiva software for a DNA experiment that uses DAPI as the DNA-staining dye.
  • Page 79 With the DNA experiment icon selected, verify in the Inspector that the Use global instrument settings checkbox is checked (Figure 3-3 on page 56). Select the Instr Settings icon in the DNA experiment, and use the Inspector to specify the parameters and threshold setting for your experiment. •...
  • Page 80 On the Population tab, deselect #Events and %Parent. • On the Statistics tab, select the mean and CV for both DAPI-A and DAPI-W, and set Decimal Places to 1 for the CVs (Figure 4-2 on page 81). BD LSR II User’s Guide...
  • Page 81 Figure 4-2 Edit Statistics view • Click OK. In the Acquisition Dashboard, set the Events to Record to 10,000 and the Events to Display to 500. Chapter 4: DNA Analysis...

  • Page 82: Running Cen

    Running CEN On the BD LSR II cytometer control panel, press RUN and LO. Install the CEN sample tube on the SIP. Verify that the green current tube pointer is in front of the CEN tube in the Browser. In the Acquisition Dashboard, click Acquire Data.
  • Page 83 Figure 4-4 Defining singlet and doublet populations CV <3% In the Acquisition Dashboard, click Record Data. When recording finishes, note the CV of the Singlet population. If the CV is ≤3%, continue to step 11. • • If the CV is >3%, restart the acquisition. Decrease the flow rate with the SAMPLE FINE ADJ knob until the CV is ≤3%, and then re-record the data.

  • Page 84: Ctn Resolution

    Discriminating the singlets from the aggregates enhances the accuracy of cell-cycle analysis. Running CTN On the BD LSR II cytometer control panel, press RUN and LO. Install the CTN sample tube on the cytometer. Adjust the event rate to approximately 500 events/second.
  • Page 85 Adjust the DAPI voltage to place the first peak at approximately channel 50 x 10 on the DAPI-A axis. Figure 4-5 Doublet discrimination with unzoomed plot singlets doublets Use the Zoom-In button to magnify the area showing the singlets and doublets on the DAPI-A vs DAPI-W plot (Figure 4-6).

  • Page 86: Optimization For Data Recording

    50 x 10 After optimizing the instrument settings, record data for each sample tube. BD FACSDiva software does not include DNA analysis algorithms. Export your data files for analysis in a third-party application such as ModFit LT™. BD LSR II User’s Guide...

  • Page 87: Chapter 5: Calcium Flux

    Calcium Flux The following topics are covered in this chapter: • Intracellular Calcium Concentration on page 88 • Calcium Flux Optimization on page 89 • Recording Calcium Flux Data on page 96...

  • Page 88: Intracellular Calcium Concentration

    (Figure 5-1). A break in data occurs when the stimulus is added to the sample tube. The increase in the ratio over time reflects the increase in intracellular Ca concentration. Figure 5-1 Calcium flux data BD LSR II User’s Guide...

  • Page 89: Calcium Flux Optimization

    Calcium Flux Optimization Before beginning this section, do the following: • Start up the instrument and perform QC. • Ensure that the appropriate filters are installed. See Setting Up the Optical Filters and Mirrors on page 39. • Review the following section, Using the Time Parameter. For calcium-flux experiments, use a sheath fluid that does not contain preservatives.

  • Page 90: Setting Up The Experiment

    For accurate data results, the Instrument Configuration dialog must reflect the physical layout of the BD LSR II octagons and trigons. Modifications to the current configuration will not apply unless you click Set Configuration. Choose Edit > User Preferences.
  • Page 91 Select the Instr Settings icon in the CalciumFlux experiment, and use the Inspector to specify parameters and settings for your experiment. • Make sure the parameters you need appear on the Parameters tab. If more than one parameter is available for a particular PMT, you might have to select the one you need from a menu.
  • Page 92 At this point, your experiment should look similar to this one. Create a normal worksheet for the CaF_001 tube with the following dot plots: • FSC-A vs SSC-A • Indo-1 (Blue)-A vs Indo-1 (Violet)-A • Time vs Ratio: Indo-1 (Violet)-A/Indo-1 (Blue)-A BD LSR II User’s Guide...
  • Page 93 Select all plots. Click the Title tab in the Inspector and select the Tubes and Populations checkboxes to display their names in plot titles. Create a statistics view and display the mean for Indo-1 (Blue)-A, Indo-1 (Violet)-A, and the ratio parameter. •...

  • Page 94: Optimizing For Calcium Flux

    FSC vs SSC dot plot. Adjust the FSC threshold to remove debris without cutting into the lymphocyte population. Draw a gate around the lymphocytes. Create a population hierarchy view and rename the population Lymphocytes. Figure 5-2 Lymphocyte gate BD LSR II User’s Guide...
  • Page 95 Display only the lymphocyte population in the remaining two dot plots. Select the two plots, right-click inside one of the plots and choose Show Populations > Lymphocytes. Adjust the Indo-1 (Violet)-A and the Indo-1 (Blue)-A voltages to optimize the signal. The signal, when displayed in the plot, should resemble that of Figure 5-3.

  • Page 96: Recording Calcium Flux Data

    If recording is stopped, ensure that the subsequent data is appended to the unstimulated sample data. When approximately 10,000 events have been recorded, remove the unstimulated sample tube from the cytometer. Add the stimulus to the tube and mix thoroughly. BD LSR II User’s Guide...
  • Page 97 In the Acquisition Dashboard, click Next Tube to create a new tube, and rename the new tube appropriately. Repeat steps 3 through 11. BD FACSDiva software does not include calcium flux analysis algorithms. Export your data files for analysis in a suitable third-party application. Chapter 5: Calcium Flux...
  • Page 98 BD LSR II User’s Guide...

  • Page 99: Chapter 6: Maintenance

    Maintenance • Daily Cleaning and Shutdown on page 100 • Scheduled Maintenance on page 103 • Periodic Maintenance on page 107...

  • Page 100: Daily Cleaning And Shutdown

    The BD LSR II 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 routine cleaning procedures that will keep your instrument in good condition.
  • Page 101 Install a tube containing 3 mL of a bleach solution on the SIP with the support arm to the side (vacuum on) and let it run for 1 minute. For the bleach solution, use BD™ FACSClean solution or a 1:10 dilution of bleach in DI water.

  • Page 102: Daily Shutdown

    Do not leave more than 1 mL of water on the SIP. When the BD LSR II flow cytometer is turned off or left in STNDBY mode, a small amount of fluid will drip back into the sample tube. If there is too much fluid in the tube, it could overflow and affect instrument performance.

  • Page 103: Scheduled Maintenance

    Perform the system flush at least every 2 weeks. Instrument hardware might be contaminated with biohazardous material. Use 10% bleach to decontaminate the BD LSR II flow cytometer. Flushing with 10% bleach is the only procedure recommended by BD Biosciences for decontaminating the instrument.
  • Page 104 Repeat steps 2 through 10 with BD FACSRinse solution in place of the bleach solution. Repeat steps 2 through 10 with DI water in place of the BD FACSRinse solution. Replace the sheath filter and refill the sheath container with sheath fluid.

  • Page 105: Waste Management System Maintenance

    Waste Management System Maintenance The waste management system for the BD LSR II instrument has an alarm powered by a 9-volt battery that must be tested and changed regularly to ensure its continued operation. Test the battery every 2 weeks after you flush the system;...
  • Page 106 Remove the battery from the drawer (Figure 6-4 on page 107). Replace a new 9-volt battery into the drawer, making sure that it is in the correct orientation. Markings in the drawer will guide you. BD LSR II User’s Guide...

  • Page 107: Periodic Maintenance

    Figure 6-4 Changing battery Slide the drawer into the bracket until you feel a click. Test the new battery. See Testing the Battery and Alarm on page 105. Periodic Maintenance The following instrument components should be checked occasionally and cleaned as necessary. The frequency will depend on how often the instrument is run.

  • Page 108: Changing The Sheath Filter

    3–6 months. Figure 6-5 Sheath filter cytometer fluid line quick-disconnect vent cap vent line quick-disconnect sheath container fluid line To avoid spraying sheath fluid, depressurize the sheath container before opening it. BD LSR II User’s Guide...
  • Page 109 Remove the Old Filter Place the instrument in STNDBY. Disconnect the air line (green). Depressurize the sheath container by lifting the vent valve. For a description of the vent valve, see Figure 2-3 on page 44. Detach the cytometer fluid line from the filter assembly by squeezing the quick-disconnect.

  • Page 110: Changing The Bal Seal

    Remove the outer droplet sleeve from the sample injection tube by turning the retainer counterclockwise. Figure 6-6 Removing the outer sleeve Bal seal Work carefully—the outer sleeve can fall out as you loosen the retainer. BD LSR II User’s Guide...
  • Page 111 Remove the Bal seal by gripping it between your thumb and index finger and pulling (Figure 6-7). Figure 6-7 Removing the Bal seal Install the new Bal seal spring-side up. Gently push the seal in place to seat it. Reinstall the retainer and outer sleeve over the sample injection tube. Tighten the retainer just enough to hold it in place.

  • Page 112: Changing The Sample Tube O-Ring

    Install a sample tube on the SIP to ensure that the outer sleeve has been properly installed. If the sleeve hits the bottom of the tube, loosen the retainer slightly and push the sleeve up as far as it will go. Tighten the retainer. BD LSR II User’s Guide...

  • Page 113: Chapter 7: Troubleshooting

    The tips in this section are designed to help you troubleshoot your experiments. You can find additional troubleshooting information in the BD FACSDiva Software Reference Manual. If additional assistance is required, contact your local BD Biosciences technical support representative. See Technical Assistance on page xv.

  • Page 114: Instrument Troubleshooting

    Waste tank full Empty the waste tank. Sample tube not fitting Sample tube other than Use BD Falcon 12 x 75-mm on SIP BD Falcon tubes used sample tubes. See Equipment on page 152. Worn Bal seal Replace the Bal seal.
  • Page 115 Threshold level too high Lower the threshold level. PMT voltage for threshold Set the PMT voltage higher for the parameter set too low threshold parameter. BD FACSDiva Gating issue Refer to the Software Reference Manual information on setting gates. Air in sheath filter Purge the filter.
  • Page 116 Sample Tube O-Ring on page 112. Bal seal worn Replace the Bal seal. See Changing the Bal Seal on page 110. Air leak at sheath container Ensure that the sheath container lid and all connectors are securely seated. BD LSR II User’s Guide...
  • Page 117 See Changing Optical Filters or Mirrors on page 42. Laser not functioning Verify laser malfunction by changing the threshold to an alternative laser while running appropriate QC particles. If not successful, contact BD Biosciences. Chapter 7: Troubleshooting...
  • Page 118 Refer to the BD FACSDiva Software Reference Manual instructions. Sample too concentrated Dilute the sample. Sample flow rate set on HI Set the sample flow rate to MED or LO. BD LSR II User’s Guide...
  • Page 119 Observation Possible Causes Recommended Solutions Low event rate Threshold level too high Lower the threshold level. Refer to BD FACSDiva Software Reference Manual instructions. PMT voltage for threshold Set the PMT voltage higher for the parameter set too low threshold parameter. Refer to the...
  • Page 120 Perform the system flush procedure. See System Flush on page 103. Air leak at sheath container Ensure that the sheath container lid is tight and all connectors are secure. Hypertonic buffers or fixative Replace the buffers and fixative. BD LSR II User’s Guide...
  • Page 121 Instrument Troubleshooting (continued) Observation Possible Causes Recommended Solutions Excessive amount of Threshold level too low Increase the threshold level. debris in display Sheath filter dirty Replace the filter. See Changing the Sheath Filter on page 108. Flow cell dirty Flush the system. See System Flush on page 103.
  • Page 122 • 30 min for the 488-nm (blue) • 30 min for the 355-nm (UV) • 15 min for the 405-nm (violet) • 20 min for the 633-nm (red) Laser not functioning Contact BD Biosciences. Optical alignment problem Contact BD Biosciences. BD LSR II User’s Guide...

  • Page 123: Appendix A: Technical Overview

    Appendix A Technical Overview This appendix contains a technical overview of the following topics: • Fluidics on page 124 • Optics on page 125 • Electronics on page 134...

  • Page 124: Fluidics

    Fluidics The fluidics system in the BD LSR II flow cytometer is pressure driven—a built-in air pump provides a sheath pressure of 5.5 psi. After passing through the sheath filter, sheath fluid is introduced into the lower chamber of the quartz flow cell.

  • Page 125: Optics

    Figure A-1 Hydrodynamic focusing of the sample core through the flow cell low sample high sample pressure pressure laser beam laser beam (12 µL/min) (60 µL/min) sheath sheath sheath sheath fluid fluid fluid fluid sample sample Optics The optics system consists of lasers, optical filters, and detectors. Lasers illuminate the cells or particles in the sample and optical filters direct the resulting light scatter and fluorescence signals to the appropriate detectors.

  • Page 126: Fluorescence

    (675 nm), while other fluorochromes such as FITC have a smaller Stokes shift, absorbing blue light and emitting green light (530 nm). The emission spectra for some commonly used fluorochromes are shown in Figure A-3 on page 127. BD LSR II User’s Guide...

  • Page 127: Optical Filters

    Figure A-3 Emission spectra of commonly used fluorochromes 100% wavelength (nm) Optical Filters Optical filters modify the spectral distribution of light scatter and fluorescence directed to the detectors. When photons encounter an optical filter, they are either transmitted, absorbed, or reflected (Figure A-4). Figure A-4 Effect of an optical filter on incident photons photons absorbed...
  • Page 128 A less steep slope indicates that more light outside the rated bandwidth is being transmitted. Two kinds of filters are used on the BD LSR II flow cytometer: • longpass (LP) •...
  • Page 129 Shortpass Filter An SP filter has the opposite properties of a longpass filter. An SP filter passes light with a shorter wavelength than the filter rating. shortpass wavelength (nm) Bandpass Filter A BP filter transmits a relatively narrow range or band of light. Bandpass filters are typically designated by two numbers.
  • Page 130 BP filter. This steep slope means that a DF filter is better at blocking light outside the rated bandwidth of the filter. BP 500/50 filter DF 500/50 filter AF 500/50 filter wavelength (nm) BD LSR II User’s Guide...

  • Page 131: Compensation Theory

    Dichroic Mirrors Dichroic filters that are used to direct different color light signals to different detectors are called dichroic mirrors or beam splitters. Although dichroic mirrors have the properties of LP or SP optical filters, you can not necessarily use any type of LP or SP filter as a beam splitter. A beam splitter must have a surface coating that reflects certain wavelengths, but many LP or SP filters are absorbance filters that do not have any specific reflective characteristics.
  • Page 132 This FITC spillover in the PE detector is to be corrected as indicated by the arrow in Figure A-7. Using the Compensation tab of the Instrument window in BD FACSDiva software, adjust the PE-%FITC spectral overlap value. BD LSR II User’s Guide...
  • Page 133 Compensation is optimal when the positive and negative FITC populations have the same means or medians in the PE parameter statistics (Figure A-8). Figure A-8 FITC spillover optimally compensated out of the PE parameter FITC positive population unstained particles means match FITC Once fluorescence compensation has been set for any sample, the compensation setting remains valid for a subsequent dim or bright sample, because...

  • Page 134: Electronics

    The peak intensity, or height of the pulse, is measured at this point. As the particle leaves the beam, the pulse trails off below the threshold. Figure A-10 Anatomy of a pulse time time time BD LSR II User’s Guide...

  • Page 135: Pulse Measurements

    Pulse Measurements The pulse processors measure pulses by three characteristics: height, area, and width (Figure A-11). Figure A-11 Pulse measurements height area threshold baseline voltage 0 volts time window gate: width • pulse height is the maximum digitized intensity measured for the pulse •...

  • Page 136: Digital Electronics

    Digital Electronics BD LSR II flow cytometer electronics digitize the signal intensity produced by a detector. The digitized data is stored in memory and further processed by the electronics to calculate • pulse height, area, and width • compensation •...

  • Page 137: Threshold

    These parameters are set by BD Biosciences service personnel when the BD LSR II flow cytometer is installed, and they rarely need to be changed. Record and save these parameter values for future reference.
  • Page 138 BD LSR II User’s Guide...

  • Page 139: Appendix B: Bd Lsr Ii Qc Log

    Appendix B BD LSR II QC Log This sample quality control (QC) log can be photocopied or used as a guide in designing your own QC log.
  • Page 140 BD LSR II User’s Guide...
  • Page 142 BD LSR II User’s Guide...

  • Page 143: Appendix C: Filter Templates

    Appendix C Filter Templates You can use these templates to note your custom filter configurations for the BD LSR II instrument. • Octagon Template on page 145 • Trigon Template on page 147...
  • Page 144 BD LSR II User’s Guide...

  • Page 145: Octagon Template

    Octagon Template Laser____________________ Experiment_____________________ Appendix C: Filter Templates...
  • Page 146 BD LSR II User’s Guide...

  • Page 147: Trigon Template

    Trigon Template Laser____________________ Experiment_____________________ Appendix C: Filter Templates...
  • Page 148 BD LSR II User’s Guide...

  • Page 149: Appendix D: Supplies And Consumables

    US, contact your local BD Biosciences customer service representative. Worldwide contact information can be found at bdbiosciences.com. Use the following part numbers to order supplies for your BD LSR II system: • QC Particles on page 150 •...

  • Page 150: Qc Particles

    • BD Biosciences • 559123 Rainbow Calibration Particles (8 peak) • all • URFP- • SPHERO Ultra • Spherotech, Inc. 30-2 Rainbow Fluorescent Particles (single peak) DNA QC Particles kit blue 488 nm BD Biosciences 349523 BD LSR II User’s Guide...

  • Page 151: Reagents

    Clorox or other major – hypochlorite) supplier (to ensure that the bleach is at the correct concentration and free of particulate matter) a. Refer to the BD Biosciences Product Catalog or the BD Biosciences website (bdbiosciences.com). Appendix D: Supplies and Consumables...

  • Page 152: Equipment

    Equipment Equipment Item Supplier Catalog No. Bal seal BD Biosciences 343509 O-ring, sample tube 343615 Sheath filter assembly 344678 BD Falcon polystyrene test tubes, BD Labware 352052 12 x 75-mm 352054 352058 BD LSR II User’s Guide...

  • Page 153: Appendix E: Standard Default Configuration

    • Standard (4-Blue 2-Violet 2-UV 2-Red) Configuration Specification on page 154 • Custom Configurations on page 159 The BD LSR II cytometer can also be ordered with one of several optional configurations, which are described in Appendix F.

  • Page 154: Standard (4-Blue 2-Violet 2-Uv 2-Red) Configuration Specification

    — blank none violet trigon 505 LP 525/50 AmCyan (405-nm laser) blank 440/40 Pacific Blue™ — blank none UV trigon 505 LP 530/30 Indo-1 (Blue) (355-nm laser) blank 450/50 Indo-1 (Violet), DAPI — blank none BD LSR II User’s Guide...

  • Page 155: Default Configuration

    Table E-1 Default filters and fluorochromes (continued) Longpass Detector Array Bandpass Fluorochrome or Dichroic (Laser) (Detector) Filter Scatter Parameter Mirror red trigon 735 LP 780/60 APC-Cy7 (633-nm laser) blank 660/20 — blank none Default Configuration Figure E-1 shows the standard default instrument configuration. Figure E-1 Default configuration Appendix E: Standard Default Configuration...

  • Page 156: Octagon And Trigon Maps

    If a slot is filled with a filter or mirror, an identifying number appears in that position on the configuration map. If a slot is filled with a blank optical holder, that position on the configuration map is unlabeled. Figure E-2 Standard default configuration: blue octagon 488-nm blue laser BD LSR II User’s Guide...
  • Page 157 Figure E-3 Standard default configuration: red and violet trigons 633-nm red laser 405-nm violet laser Appendix E: Standard Default Configuration...
  • Page 158 Figure E-4 Standard default configuration: UV trigon 355-nm UV laser BD LSR II User’s Guide...

  • Page 159: Custom Configurations

    Table E-2 shows the detector arrays, mirrors, and filters used in the custom configurations, and recommended fluorochromes per detector. The mirrors and filters used in these custom configurations are contained in the BD LSR II instrument spares kit. Table E-2 Spare filters and fluorochromes...
  • Page 160 PE-Texas Red™ To use PE-Texas Red™, replace the mirror and filter for the B PMT of the blue octagon as shown below. 488-nm blue laser BD LSR II User’s Guide...
  • Page 161 Indo-1 If you have a violet laser, replace the mirror for the A PMT of the UV trigon as shown on the left below. If you do not have a violet laser, replace both the mirror for the A PMT and the filter for the B PMT of the UV trigon as shown on the right below.
  • Page 162 DsRed To use DsRed, replace the filter for the C PMT of the blue octagon as shown below. 488-nm blue laser BD LSR II User’s Guide...
  • Page 163 PerCP or BD Cy-Chrome Reagent To use PerCP or BD Cy-Chrome reagent, replace the mirror and filter for the B PMT of the blue octagon as shown below. 488-nm blue laser Appendix E: Standard Default Configuration...
  • Page 164 BD LSR II User’s Guide...

  • Page 165: Appendix F: Special Order Configurations

    Appendix F Special Order Configurations The BD LSR II cytometer can be ordered with, or upgraded to one of several laser and detector array options. If you upgrade your configuration, you must use BD FACS Instrument Configuration Application (ICA) to update your BD FACSDiva software default instrument configuration (database object).

  • Page 166: Common Special Order Configurations

    6-Blue 6-Violet 0-UV 3-Red Configuration on page 172 • 6-Blue 6-Violet 0-UV 4-Red Configuration on page 174 • 6-Blue 6-Violet 2-UV 3-Red Configuration on page 176 • 6-Blue 6-Violet 2-UV 4-Red Configuration on page 178 BD LSR II User’s Guide...

  • Page 167: 6-Blue 0-Violet 0-Uv 3-Red Configuration

    6-Blue 0-Violet 0-UV 3-Red Configuration 6-Blue 0-Violet 0-UV 3-Red supports a blue octagon, and a red trigon. Table F-1 shows the detectors, filters, and mirrors used in the default configuration. The word “blank” indicates that a blank optical holder should be used instead of a mirror or filter.

  • Page 168: 6-Blue 2-Violet 0-Uv 3-Red Configuration

    755 LP 780/60 PE-Cy7 (488-nm laser) 685 LP 695/40 PerCP-Cy5.5 655 LP 660/20 PE-Cy5 600 LP 610/20 PE-Texas Red™ 550 LP 575/26 505 LP 530/30 FITC, Alexa Fluor® 488 blank 488/10 — blank none BD LSR II User’s Guide...

  • Page 169: 6-Blue 0-Violet 2-Uv 3-Red Configuration

    Table F-2 6-Blue 2-Violet 0-UV 3-Red default mirror and filter configuration (continued) Longpass Detector Array Bandpass Fluorochrome or Dichroic (Laser) (Detector) Filter Scatter Parameter Mirror violet trigon 505 LP 525/50 AmCyan (405-nm laser) blank 450/50 Pacific Blue™ — blank none red trigon 755 LP 780/60...
  • Page 170 UV trigon 505 LP 530/30 Indo-1 (Blue) (355-nm laser) blank 450/50 Indo-1 (Violet), DAPI — blank none red trigon 755 LP 780/60 APC-Cy7 (633-nm laser) 710 LP 730/45 Alexa Fluor® 700 — 660/20 BD LSR II User’s Guide...

  • Page 171: 6-Blue 2-Violet 2-Uv 3-Red Configuration

    6-Blue 2-Violet 2-UV 3-Red Configuration 6-Blue 2-Violet 2-UV 3-Red supports a blue octagon, and violet, UV, and red trigons. Table F-4 shows the detectors, filters, and mirrors used in the default configuration. The word “blank” indicates that a blank optical holder should be used instead of a mirror or filter.

  • Page 172: 6-Blue 6-Violet 0-Uv 3-Red Configuration

    The 6-Blue 6-Violet 0-UV 3-Red maps are: • 6-Color Blue Octagon Default Configuration Map on page 181 • 6-Color Violet Octagon Default Configuration Map on page 183 • 3-Color Red Trigon Default Configuration Map on page 185 BD LSR II User’s Guide...
  • Page 173 Table F-5 6-Blue 6-Violet 0-UV 3-Red default mirror and filter configuration Longpass Detector Array Bandpass Fluorochrome or Dichroic (Laser) (Detector) Filter Scatter Parameter Mirror blue octagon 755 LP 780/60 PE-Cy7 (488-nm laser) 685 LP 695/40 PerCP-Cy5.5 655 LP 660/20 PE-Cy5 600 LP 610/20 PE-Texas Red™...

  • Page 174: 6-Blue 6-Violet 0-Uv 4-Red Configuration

    755 LP 780/60 PE-Cy7 (488-nm laser) 685 LP 695/40 PerCP-Cy5.5 655 LP 660/20 PE-Cy5 600 LP 610/20 PE-Texas Red™ 550 LP 575/26 505 LP 530/30 FITC, Alexa Fluor® 488 blank 488/10 — blank none BD LSR II User’s Guide...
  • Page 175 Table F-6 6-Blue 6-Violet 0-UV 4-Red default mirror and filter configuration (continued) Longpass Detector Array Bandpass Fluorochrome or Dichroic (Laser) (Detector) Filter Scatter Parameter Mirror violet octagon 630 LP 655/8 Qdot 655 (405-nm laser) 595 LP 605/12 Qdot 605 575 LP 585/15 Qdot 585 545 LP...

  • Page 176: 6-Blue 6-Violet 2-Uv 3-Red Configuration

    755 LP 780/60 PE-Cy7 (488-nm laser) 685 LP 695/40 PerCP-Cy5.5 655 LP 660/20 PE-Cy5 600 LP 610/20 PE-Texas Red™ 550 LP 575/26 505 LP 530/30 FITC, Alexa Fluor® 488 blank 488/10 — blank none BD LSR II User’s Guide...
  • Page 177 Table F-7 6-Blue 6-Violet 2-UV 3-Red default mirror and filter configuration (continued) violet octagon 630 LP 655/8 Qdot 655 (405-nm laser) 595 LP 605/12 Qdot 605 575 LP 585/15 Qdot 585 545 LP 560/20 Qdot 565 475 LP 525/50 AmCyan, Qdot 525 blank 450/50 Pacific Blue™...

  • Page 178: 6-Blue 6-Violet 2-Uv 4-Red Configuration

    755 LP 780/60 PE-Cy7 (488-nm laser) 685 LP 695/40 PerCP-Cy5.5 655 LP 660/20 PE-Cy5 600 LP 610/20 PE-Texas Red™ 550 LP 575/26 505 LP 530/30 FITC, Alexa Fluor® 488 blank 488/10 — blank none BD LSR II User’s Guide...
  • Page 179 Table F-8 6-Blue 6-Violet 2-UV 4-Red default mirror and filter configuration (continued) Longpass Detector Array Bandpass Fluorochrome or Dichroic (Laser) (Detector) Filter Scatter Parameter Mirror violet octagon 630 LP 655/8 Qdot 655 (405-nm laser) 595 LP 605/12 Qdot 605 575 LP 585/15 Qdot 585 545 LP...

  • Page 180: Special Order Configuration Trigon And Octagon Maps

    6-Color Violet Octagon Default Configuration Map on page 183 • 2-Color UV Trigon Default Configuration Map on page 184 • 3-Color Red Trigon Default Configuration Map on page 185 • 4-Color Red Octagon Default Configuration Map on page 186 BD LSR II User’s Guide...

  • Page 181: 6-Color Blue Octagon Default Configuration Map

    6-Color Blue Octagon Default Configuration Map 488-nm blue laser Appendix F: Special Order Configurations...

  • Page 182: 2-Color Violet Trigon Default Configuration Map

    2-Color Violet Trigon Default Configuration Map 405-nm violet laser BD LSR II User’s Guide...

  • Page 183: 6-Color Violet Octagon Default Configuration Map

    6-Color Violet Octagon Default Configuration Map 405-nm violet laser Appendix F: Special Order Configurations...

  • Page 184: 2-Color Uv Trigon Default Configuration Map

    2-Color UV Trigon Default Configuration Map 355-nm UV laser BD LSR II User’s Guide...

  • Page 185: 3-Color Red Trigon Default Configuration Map

    3-Color Red Trigon Default Configuration Map 633-nm red laser Appendix F: Special Order Configurations...

  • Page 186: 4-Color Red Octagon Default Configuration Map

    4-Color Red Octagon Default Configuration Map 633-nm red laser BD LSR II User’s Guide...

  • Page 187: Bd Facs Instrument Configuration Application

    • Updating Your Default Configuration on page 188 Installing ICA To install the BD FACS Instrument Configuration Application: Mount your ICA CD on an available drive. If the installer setup program does not start automatically, launch setup.exe from the CD root folder. After the setup runs, the InstallShield Wizard will lead you through several installation screens.

  • Page 188: Updating Your Default Configuration

    ICA activity window as it would appear after step 3 below). Choose LSRII from the Select Instrument pull-down menu. The Select Configuration scroll view lists the available BD LSR II instrument configurations. Select the BD LSR II instrument configuration that matches your configuration upgrade.
  • Page 189 Figure F-1 ICA: Select Configuration field Appendix F: Special Order Configurations...
  • Page 190 Figure F-2 ICA: upgrade log BD LSR II User’s Guide...
  • Page 191 Click Save Log to save a file containing the current text of the ICA main text view. A Save As dialog is displayed (Figure F-3). Choose a location and name for your log file, and click Save. Figure F-3 Select configuration Click Exit to exit the program.
  • Page 192 BD LSR II User’s Guide...

  • Page 193: Appendix G: Setting Laser Delay

    Appendix G Setting Laser Delay This appendix describes how to optimize laser delay settings in a multiple laser system. • About Laser Delay on page 194 • Optimizing Laser Delay on page 195...

  • Page 194: About Laser Delay

    This allows optimal detection of fluorescent signal from each laser with minimal cross-contamination from the other beams. In the BD LSR II four-laser system, the blue laser intercepts the stream first, followed by the violet, UV, and red lasers. Because the laser signals are spatially separated, there is a slight delay between the detection of each laser’s signal...

  • Page 195: Optimizing Laser Delay

    Optimizing Laser Delay Laser delay is set using BD FACSDiva software. To optimize the delay for a given laser, you acquire events from a sample with a fluorescence signal excited by that laser. Follow the procedures in Chapter 3, Running Samples, for sample optimization and acquiring data.
  • Page 196 6. You should also stay within a range of 10 µsec of the initial setting (step 5). Preserve the setting that maximizes the fluorescence intensity. Reset the window extension to 10 µsec. BD LSR II User’s Guide...
  • Page 197 BD FACS Instrument Configuration threshold 60 Application (ICA) 189 voltages 60 BD FACSClean solution 103 air in filter, removing 47, 48 BD FACSDiva software See software alarm, waste container 31 BD FACSFlow solution 123 ALPHA filters (AFs) 34 BD FACSRinse solution 103 analysis...
  • Page 198 36 configuration maps 158 dichroic containers, sheath and waste 31, 44, 47 filters 34 control panel, instrument 27 mirrors 34, 133 controls digital data 26 compensation 59, 60 discriminating filters (DFs) 132 single-stained 54, 66 BD LSR II User’s Guide...
  • Page 199 filters experiment criteria 78 bandpass 34 flow rate for analysis 126 optical QC Particles kit 79 bandpass 34, 131 using DAPI 79 changing 42 verifying linearity 78 dichroic 34 doublet discrimination 79, 84 discriminating 132 droplet containment system 30 extra 162 troubleshooting 116 longpass 34, 130 DsRed, filter configuration for 165...
  • Page 200 164 longpass dichroic mirrors instrument holder 40 components shown 26 specifications 43 configuration 41, 54 LSR II See instrument, BD LSR II. covers and door 26, 42 dimensions xiii experiment criteria 78 handles 27, 32 BD LSR II User’s Guide...
  • Page 201 102 DsRed 165 flushing system 105 indo-1 164 periodic PerCP or BD Cy-Chrome 166 Bal seal 112 PE-Texas Red 163 sample tube O-ring 114 dichroic mirrors 34, 133 sheath filter 110, 111 filters 34, 42, 129...
  • Page 202 (SSC) 33, 127 signals, amplifying 35 singlet population, discrimination 84 safety SIP See sample injection port. biological xx software electrical xix adjusting detector voltages 36 general xxi instrument control xi laser xvii symbols and labels xxi BD LSR II User’s Guide...
  • Page 203 spares filters and mirrors 162 ungrounded receptacles xix kit 162 Use global instrument settings 57 parts, ordering 152 user preferences 56, 80, 92 specifications, filter and mirror 43 UV laser 33 specimen Calcium Flux setup 94 CEN optimization 82 spillover 133 violet laser 33 statistics views 70 virus protection software xxiii...
  • Page 204 BD LSR II User’s Guide...

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