Dead cell discrimination with 7-amino-actinomcin D in combination with dual color immunofluorescence in single laser flow cytometry

Authors

  • Ingrid Schmid,

    Corresponding author
    1. Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024
    2. Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California 90024
    • UCLA School of Medicine, Department of Medicine/Cellular Immunology and Cytometry, 12–244 Factor Building Los Angeles, CA 90024-1745
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  • Wanda J. Krall,

    1. Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California 90024
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  • Christel H. Uittenbogaart,

    1. Department of Pediatrics, UCLA School of Medicine, Los Angeles, California 90024
    2. Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California 90024
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  • Jonathan Braun,

    1. Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California 90024
    2. Department of Pathology, UCLA School of Medicine, Los Angeles, California 90024
    3. Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California 90024
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  • Janis V. Giorgi

    1. Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024
    2. Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California 90024
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  • This work was performed in the UCLA Jonsson Comprehensive Cancer Center Flow Cytometry Laboratory and was supported by National Institutes of Health award CA-16042 and the McCarthy Fund for Cancer Research. W. J. K. was supported by NIH pre-doctoral training grant GM-07185.

Abstract

Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non-vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7-amino-actinomycin D (7-AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7-AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7-AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)-fluorescence dual-color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7-AAD was used on fluoresceinisothiocyanate (FITC) and PE surface-labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.

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