Flow cytometric titration of retroviral expression vectors: Comparison of methods for analysis of immunofluorescence histograms derived from cells expressing low antigen levels

Authors

  • Todd L. Sladek,

    1. Department of Genetics, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106
    Search for more papers by this author
  • Dr. James W. Jacobberger

    Corresponding author
    1. Department of Genetics, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106
    • Department of Genetics, Case Western Reserve University, School of Medicine, 2109 Adelbert Road, Cleveland, OH 44106-4955
    Search for more papers by this author

  • This work was supported by grants HL41945 and CA43703 from the National Institutes of Health and by a grant from the Diabetes Association of Greater Cleveland

Abstract

Few quantitative studies addressing immunofluorescence histogram analysis have been published. One study by Overton (Cytometry 9:619–626, 1988) has shown threshold and histogram subtraction methods to be accurate for analysis of well-separated immunofluorescence distributions of positive and negative cells. An evaluation of methods to analyze immunofluorescence histograms when positive and negative immunofluorescence distributions overlap has not, to our knowledge, been reported. In this paper, data obtained from flow cytometry of immunofluorescently stained cells infected with recombinant retroviruses that produce a range of simian virus 40 large T antigen levels were analyzed by threshold, histogram subtraction, and distribution modeling methods. This analysis showed that as the separation between the immunofluorescence distributions of positive and negative cell populations decrease the best methods for histogram analysis are modeling followed, in order, by histogram subtraction, and threshold analysis. © 1993 Wiley-Liss, Inc.

Ancillary