Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation

Authors

  • Ken Toba,

    1. Departments of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322
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  • Elliott F. Winton M.D.,

    Corresponding author
    1. Departments of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322
    • Leukemia Research Laboratory, 4000 Winship Cancer Center, 1327 Clifton Road, N. E., Atlanta, GA 30322
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  • Robert A. Bray

    1. Departments of Pathology and the Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322
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  • This work was supported by the Jill Andrews Memorial Research Fund

Abstract

We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (> 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.

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