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Flow Cytometry Analysis Using the Becton Dickinson FACS Calibur

Kevin L. Holmes1,  Gillis Otten2,  Wayne M. Yokoyama3

1National Institute of Allergy and Infectious Disease, Bethesda, Maryland
2Cell Genesys Inc., Foster City, California
3Washington University School of Medicine, St. Louis, Missouri



Publication Name: 
Current Protocols in Immunology
Unit Number: 
UNIT 5.4
DOI: 
10.1002/0471142735.im0504s49
Online Posting Date: 
August, 2002
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Abstract

This unit details the operation of a FACS Calibur flow cytometer for cell analysis. The operation of the Becton Dickinson FACS Calibur and CELLQuest software version 3.0 is described, but the unit is general enough to be helpful for users of all flow cytometers. The FACS Calibur replaces both the FACSCan and FACSort; the information presented here is also applicable to older BD instruments. In this unit, particular emphasis is placed on data acquisition rather than data analysis. Single-color analysis using fluorescein isothiocyanate (FITC)-conjugated antibodies is described along with procedures to check instrument performance and sensitivity, single-color (FITC) analysis with simultaneous live/dead discrimination using propidium iodide (PI), simultaneous two-color analysis using FITC- and phycoerythrin (PE)-conjugated antibodies, two-color FITC/PE analysis with simultaneous live/dead discrimination using PI, simultaneous three-color immunofluorescence with FITC, PE, and the red fluorescent dyes, and finally, simultaneous four-color immunofluorescence with FITC, PE, red fluorescence dyes, and APC.

     
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Table of Contents

  • Unit Introduction
  • Description of the FACS Calibur/CELLQuest System
  • Basic Protocol: Single-Color Analysis Using FITC-Conjugated Antibodies
  • Support Protocol 1: Instrument Checkout Using Calibrite Beads and FACSComp Software
  • Support Protocol 2: Discrimination of Live/Dead Cells in Single-Color Analysis
  • Alternate Protocol 1: Two-Color Analysis Using FITC- and PE-Conjugated Antibodies
  • Support Protocol 3: Discrimination of Live/Dead Cells in Two-Color Analysis
  • Alternate Protocol 2: Three-Color Analysis
  • Alternate Protocol 3: Four-Color Analysis
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
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Materials

Basic Protocol: Single-Color Analysis Using FITC-Conjugated Antibodies

 Materials
  • Isotonic saline for FACS Calibur sheath solution reservoir (follow Becton Dickinson's recommendations for source)
  • Control and test cell populations: 1–2 × 106 cells/ml unstained control cells, FITC-stained positive control cells, and FITC-stained test cell samples (unit 5.3), kept on ice in sample buffer (³250 µl each)
  • Sample buffer: 2% (v/v) heat-inactivated FBS or 1% (w/v) BSA/0.1% (w/v) sodium azide (either solution prepared in PBS)
  • 10% (v/v) bleach (sodium hypochlorite; Clorox) in deionized water
  • Flow cytometry system consisting of:
  •     FACS Calibur (Becton Dickinson)
  •     FACStation acquisition interface for Macintosh (Becton Dickinson)
  •     Macintosh Quadra 650 (or higher Quadra series) computer with 16 MB RAM and 1 MB VRAM
  •     Macintosh system software version 7 or higher
  •     Macintosh compatible printer

NOTE: Version 1.0 of CELLQuest does not support PowerMac computers for data acquisition.

Support Protocol 1: Instrument Checkout Using Calibrite Beads and FACSComp Software

 Additional Materials (also see Basic Protocol)
  • CaliBRITE standard fluorescent beads (Becton Dickinson)
  • FACSComp software program (Becton Dickinson)

Support Protocol 2: Discrimination of Live/Dead Cells in Single-Color Analysis

 Additional Materials (also see Basic Protocol)
  • 100 µg/ml propidium iodide (PI) in PBS: store at 4°C protected from light

CAUTION: PI is a potential carcinogen and mutagen and should be handled with extreme care.

Alternate Protocol 1: Two-Color Analysis Using FITC- and PE-Conjugated Antibodies

 Additional Materials (also see Basic Protocol)
  • Control and test cell populations: 1–2 × 106 cells/ml unstained control cells, FITC- and PE-stained positive control cells, and test cell samples stained with FITC and/or PE-conjugated antibodies (unit 5.3), kept on ice in sample buffer (³250 µl each; see Basic Protocol)

Support Protocol 3: Discrimination of Live/Dead Cells in Two-Color Analysis

 Additional Materials (also see Basic Protocol and Alternate Protocol 1)
  • 100 µg/ml propidium iodide (PI) in PBS: store at 4°C protected from light

CAUTION: PI is a potential carcinogen and mutagen; handle with extreme care .

Alternate Protocol 2: Three-Color Analysis

 Additional Materials (also see Basic Protocol and Alternate Protocol 1)
  • Red-stained positive control cells (see commercially available red fluorchromes in Table 5.4.1)
  • Test cell samples stained with one to three fluorochrome-conjugated antibody combinations (FITC, PE, red)
 
Table 5.4.1 Fluorochromes Used with the FACS Calibur
Fluorochromesa, bEmission peak (nm)DetectorFilter
Fluorescein isothiocyanate (FITC)525 (green)FL1530/30
R-phycoerythrin (PE)575 (orange-red)FL2585/42
Propidium iodide (PI)620 (red)FL2, FL3
PerCP675 (red)FL3670LP
PE-Cy7b767 (red)FL3
PE-Cy5b670 (red)FL3
APC660FL4661/16
Cy-5670FL4
 aDyes that cannot be used with the FACS Calibur are rhodamine and its derivatives, as these are not excited by the 488-nm argon ion laser. Texas red is very poorly excited at 488 nm and other orange and red fluorescent dyes listed above are much preferred.
 bPE-Cy5 also known as CyChrome (Pharmingen), TriColor (Caltag), Quantum Red (Sigma), and RED670 (Life Technologies).

Alternate Protocol 3: Four-Color Analysis

 Additional Materials (also see Basic Protocol and Alternate Protocol 2)
  • APC-labeled beads (Becton Dickinson)
  • APC (or Cy-5) -stained positive control cells and PE-Cy5 (or PerCP) -stained positive control cells
  • Test cell samples stained with one to four fluorochrome-conjugated antibody combinations (i.e., FITC, PE, PE-CY5 and/or APC)
Looking for materials?  Suppliers Guide
     
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Figures

  • Figure 5.4.1
    Dot density plot displays of (A) murine spleen cells and (B) human peripheral blood leukocytes. (A) Region including leukocytes and excluding red blood cells and dead cells is shown. (B) Regions including lymphocytes, monocytes or granulocytes cell populations.

    View Image
  • Figure 5.4.2
    Two-color density-plot displays and setting of FITC/PI compensation. (A) Unstained and FITC-stained cells without electronic fluorescence compensation. (B) Same cells after the FL2 – %FL1 compensation has been set. (C) Unstained cells incubated with PI. Live cells appear below the horizontal line, while dead cells appear above. (D) FITC-stained cells and unstained cells in the presence of PI. Live cells appear below the horizontal line, while dead cells that are both FITC+ and FITC– appear above; therefore, a region defined below the horizontal line includes live cells and excludes dead ones.

    View Image
  • Figure 5.4.3
    FL1 versus FL2 density plot displays used to optimize fluorescence compensation for the analysis of double- (i.e., FITC/PE-) stained cells. (A) Unstained and FITC-stained cells without compensation. (B) Same cells as (A) after FL2 – %FL1 compensation. (C) Same cells as (B) with overcompensation. (D) Unstained and PE-stained cells without compensation. (E) Same cells as (D) after FL1 – %FL2 compensation. (F) Same cells as (E) with overcompensation.

    View Image

Literature Cited

 Literature Cited
    Cooper, H.M. and Paterson, Y. 1993. Determination of the specific antibody titer. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 11.16.1-11.16.13. John Wiley & Sons, New York.
    Shapiro, H.M. 1995. Practical Flow Cytometry, 3rd ed. Wiley-Liss, New York.
 Key References
    (Darzynkiewicz, Z., Robinson, J.P., and Crissman, H.A. eds.) 1994. Flow Cytometry, 2nd ed. Methods Cell Biol.:41 & 42. Academic Press, San Diego.

Provides detailed descriptions of a wide variety of flow cytometry protocols and techniques, with extensive references.

    FACS Calibur User's Guide, CELLQuest Software User's Guide, and FACS Calibur FACSComp User's Guide. Becton Dickinson, San Jose, Calif. (see appendix 5)

These manuals offer a complete description and guide to use of the hardware and software detailed in this unit.

    Fluorescent Microbead Standards. 1988. Flow Cytometry Standards, Research Triangle Park, N.C.

Excellent monograph that provides details about calibration as well as general information about flow cytometry.

    Parks, D.R., Herzenberg, L.A., and Herzenberg, L.A. 1989. Flow cytometry and fluorescence-activated cell sorting. In Fundamental Immunology (W.E. Paul, ed.) pp. 781-802. Raven Press, New York.

Provides an excellent introduction to flow cytometry and multi-parameter analysis, with thorough discussion of multi-parameter data analysis, including data display techniques and statistical analysis, and examples of multi-color immunofluorescence data.

    Shapiro, H.M. 1995. See above.

Details all aspects of flow cytometry.

     
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