A Software Method for Color Compensation

Carleton C. Stewart1, Sigrid J. Stewart1

1 Roswell Park Cancer Institute, Buffalo, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 10.15
DOI:  10.1002/0471142956.cy1015s23
Online Posting Date:  February, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

When two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome. Spectral compensation is the process of removing the undesired overlap of signal. Although very successful for two and three fluorochromes, the general practice of adjusting instrument compensation becomes increasingly inadequate and unforgiving as the number of fluorochromes increases. When data are collected uncompensated, software compensation provides the flexibility of setting correct compensation every time for every sample. Software methods do have problems. The linearization assumptions made by the software algorithms may be more or less in error. Binning effects become more of a problem with increasing numbers of compensated parameters. This explanatory unit also contains protocols that illustrate the process of software compensation utilizing matrix algebra that provides for elements of all possible PMT detection combinations. Although details are limited to four colors, the principles described can be applied to any desired number. When two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome.

Keywords: flow cytometry; spectral compensation; software compensation; tandem dyes

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Acquisition of Data from Compensation Standards
  • Correction of Compensation for all Nontandem Fluorochromes
  • Verification of Compensation
  • Generate Compensation Matrices for each Antibody Combination
  • Data from Specimens
  • Using a Macro
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   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.
   Shapiro, H.M. 1995. Practical Flow Cytometry. 3rd ed pp.160‐162. Wiley‐Liss, New York.
   Stewart, C.C. and Mayers, G.L. 2000. Kinetics of antibody binding to cells. In Immunophenotyping, (C.C. Stewart and J. Nicholson, eds.) pp 1‐21. J. Wiley & Sons, Inc., New York.
   Stewart, C.C. and Stewart, S.J. 1999. Four color compensation. Comm. Clin. Cytometry 38:161‐175.
   Stewart, C.C. and Stewart, S.J. 2001a. Cell preparation for the identification of leukocytes. In Methods in Cell Biology, vol. 64 (Z. Darzynkiewicz, H. Crissman, and J.P. Robinson, eds.) pp. 218‐270. Academic Press, New York.
   Stewart, C.C. and Stewart, S.J. 2001b. Multiparameter data acquisition and analysis of leukocytes by flow cytometry. In Methods in Cell Biology, vol. 64 (Z. Darzynkiewicz, H. Crissman, and J.P. Robinson, eds.) pp. 289‐312. Academic Press, New York.
   van Vugt, M.J., van den Herik‐Oudijk, I.E., and van de Winkle, J.G. 1996. Binding of PE‐CY5 conjugates to the human high‐affinity receptor for IgG (CD64). Blood 88:2358‐2361.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library