Compensation in Flow Cytometry

Mario Roederer1

1 Vaccine Research Center NALID, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.14
DOI:  10.1002/0471142956.cy0114s22
Online Posting Date:  December, 2002
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Abstract

Many people using multi‐color flow cytometry do not understand what compensation is, when it is needed, or how it should be applied. The author provides a clear explanation of the nature of compensation, the factors that affect compensation values, and the effect of compensation on data visualization. Both hardware and software compensation are covered. Although the subject is treated from a mathematical perspective, the manner of presentation is very straightforward. The unit discusses appropriate controls and protocols to check compensation settings, as well as introducing the concept of fluorescence‐minus‐one gates. Many example figures are supplied to give the reader a clear indication of how the author believes compensation should be handled. Keywords: flow cytometry; spectral compensation; fluorescence spillover; autofluorescence; fluorescence minus one

     
 
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Table of Contents

  • Basic Compensation
  • Pairwise Compensation on Flow Cytometers: Three‐Color Compensation
  • Complete Compensation
  • Autofluorescence
  • Resonance Energy Transfer (Tandem) Dyes
  • Compensation: Effect on Visualization of Data
  • Gating and Analysis Controls: FMO Controls
  • Compensation Controls: Cells
  • Compensation Controls: Beads
  • Factors Affecting Compensation Values
  • Compansation Myths
  • The Protocol: How to Set Compensation
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

Videos

Literature Cited

Literature Cited
   Alberti, S., Parks, D.R., and Herzenberg, L.A. 1987. A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry 8:114‐119.
   Bagwell, C.B. and Adams, E.G. 1993. Fluorescence spectral overlap compensation for any number of flow cytometry parameters. Ann. N.Y. Acad. Sci. 677:167‐184.
   Kantor, A. and Roederer, M., 1996. FACS analysis of leukocytes. In Handbook of Experimental Immunology, 5 th ed. (L.A. Herzenberg, D.M. Weir, L.A. Herzenberg, and C. Blackwell, eds.) pp. 49.1‐49.13. Blackwell Scientific, Cambridge.
   Loken, M.R., Parks, D.R., and Herzenberg, L.A. 1977. Two‐color immunofluorescence using a fluorescence‐activated cell sorter. J. Histochem. Cytochem. 25:899‐907.
   Roederer, M. and Murphy, R.F. 1986. Cell‐by‐cell autofluorescence correction for low signal‐to‐noise systems: Application to epidermal growth factor endocytosis by 3T3 fibroblasts. Cytometry 7:558‐565.
   Roederer, M. 2001. Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats. Cytometry 45:194‐205.
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