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Analysis of Flow Cytometry Data

Susan O. Sharrow¹

¹National 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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Abstract

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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Unit Introduction
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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Figures

Figure 5.2.1

Single-parameter fluorescence histograms. Increasing fluorescence intensity is plotted on the x axis in log fluorescence units versus cell number on the y axis. Maximum values on the y axis have been adjusted to a constant percentage of total cells. This adjustment normalizes the area under the curves to a constant total area, regardless of the total cell number analyzed. Cells were reacted with positive FITC-monoclonal antibodies (1,2) or with negative-control FITC- monoclonal antibody (3). In A and B, the dashed line drawn at the x-axis value defines the region of specifically positive cells (the lower limit of interest). In C, all cells are clearly positive and frequency analysis is not appropriate.

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Figure 5.2.2

Dual-parameter contour plots. Increasing green fluorescence intensity (Ab1) is plotted on the x axis in channel numbers versus increasing red fluorescence intensity (Ab2) on the y axis. Values of the percentage of total cells were selected on the z axis (cell number) to draw the rings or contours around peaks of cells correlating green and red fluorescence. The z-axis levels chosen were 0.01% (A and B), 0.05% (C), and 0.002% (D) of total cell number. Areas of interest are indicated by boxes which were chosen using the display shown in D. Cells were stained with either positive FITC-Ab1 (A,C,D) or negative FITC-Ab (B), followed by positive Bio-Ab plus texas red–avidin (all). The same data file is displayed with three different choices of z-axis values (A,C,D). The percentage values within the boxes are the result of calculation of the percentage of all cells within that region of interest.

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Figure 5.2.3

Calibration of logarithmic amplifier. Six fluorescent-bead standards (A, B, C, D, E, and F) containing different numbers of molecules of the same fluorochrome were analyzed by flow cytometry using constant instrument variables and a nominally 3-decade logarithmic amplifier. The precalibrated value of fluorochrome-molecule number is shown (fluorochrome equivalents). Data obtained from each particle were stored and the median fluorescence channel number calculated from the histogram obtained for each particle.

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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.