Analysis of Flow Cytometry Data

Susan O. Sharrow1

1 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.2
DOI:  10.1002/0471142735.im0502s00
Online Posting Date:  May, 2001
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This unit provides several approaches to flow cytometry data analysis. Frequency determinations based on analysis of single‐parameter fluorescence histograms and dual‐parameter contour plots are presented. Next, steps are described for calculating values for signal‐to‐noise ratios when logarithmic amplification is used for data collection. This calculation can be used to compare the amounts of antigenic determinants per cell between different cells stained with the same reagent. Finally, a procedure is described for constructing a calibration curve for logarithmic amplifiers. This calibration curve is required for calculation of signal‐to‐noise values and can also be used to determine if the amplifiers are working properly.

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Table of Contents

  • Frequency Determinations Using Single‐Parameter Fluorescence Histograms
  • Frequency Determinations Using Dual‐Parameter Contour Plots
  • Comparison of Relative Signal‐to‐Noise Values
  • Construction of a Calibration Curve for a Logarithmic Amplifier
  • Critical Parameters
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
   Dower, S.K., Ozato, K., and Segal, D.M. 1984. The interaction of monoclonal antibodies with MHC class I antigens on mouse spleen cells. I. Analysis of the mechanism of binding. J. Immunol. 132:751‐758.
   Flow Cytometry Standards, 1988. Fluorescent Microbead Standards (Monograph). Flow Cytometry Standards, Research Triangle Park, N. C.
   Gandler, W. and Shapiro, H. 1990. Technical tutorial: Logarithmic amplifiers. Cytometry 11:447‐450.
   Ledbetter, J.A., Evans, R.L., Lipinski, M., Cunningham‐Rundles, C., Good, R.A., and Herzenberg, L.A. 1981. Evolutionary conservation of surface molecules that distinguish T lymphoyte helper/inducer and cytotoxic/suppressor subpopulations in mouse and man. J. Exp. Med. 153:310‐323.
   Mason, D.W. and Williams, A.F. 1980. The kinetics of antibody binding to membrane antigens in solution and at the cell surface. Biochem. J. 187:1‐9.
   Mathieson, B.J., Sharrow, S.O., Campbell, P.S., and Asofsky, R. 1979. An Lyt differentiated thymocyte subpopulation detected by flow microfluorometry. Nature (Lond.). 277:478‐480.
   Mathieson, B.J., Sharrow, S.O., Bottomly, K., and Fowlkes, B.J. 1980. Ly‐9: An alloantigenic marker of lymphocyte differentiation. J. Immunol. 125:2263‐2268.
   Parks, D.R., Bigos, M., and Moore, W.A. 1988. Logarithmic amplifier transfer function evaluation and procedures for logamp optimization and data correction. Cytometry (Suppl.) 2:27.
   Parks, D.R., Herzenberg, L.A., and Herzenberg, L.A. 1989. Flow cytometry and fluorescence activated cell sorting. In. Fundamental Immunology, 2nd ed (W. Paul, ed.) pp. 803‐818. Raven Press, New York.
   Sanders, M.E., Makgoba, M.W., Sharrow, S.O., Stephany, D., Springer, T.A., Young, H.A., and Shaw, S. 1988. Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA‐3, CD2, and LFA‐l) and three other molecules (UCHLl, CDw29, and Pgp‐l) and have enhanced IFN‐gamma production. J. Immunol. 140:1401‐1407.
   Schmid, I., Schmid, P., and Giorgi, J.V. 1988. Conversion of logarithmic channel numbers into relative fluorescence intensity. Cytometry 9:533‐538.
   Segal, D.M., Titus, J.A., and Stephany, D.A. 1987. Fluorescence flow cytometry in the study of lymphoid cell receptors. Methods Enzymol. 150:478‐492.
   Titus, J.A., Haugland, R., Sharrow, S.O., and Segal, D.M. 1982. Texas red, a hydrophilic, red‐emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J. Immunol. Methods 50:193‐204.
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