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Estimation of Cell Survival by Flow Cytometric Quantification of Fluorescein Diacetate/Propidium Iodide Viable Cell Number¹

Program of Oncology, University of Maryland Cancer Center [D. D. R., C. C. J., J. V. O., A. M. S., R. K. W., A. W. H., R. E. N.], the Department of Medicine, Division of Hematology [D. D. R.], and the Department of Pathology [A. W. H.], University of Maryland School of Medicine, Baltimore, Maryland

We report a flow cytometric method to quantify the number of viable cells remaining in suspension culture following exposure to cytotoxic drugs. Cell viability is assessed by flow cytometric measurement of cellular fluorescence after staining with fluorescein diacetate and propidium iodide in isotonic solution. The number of viable cells per ml of culture is determined by a timed count of viable cells and from knowledge of the flow cytometer sample flow rate. P388 murine or HL-60 human leukemia cells in culture were used as model systems. This method can quantify accurately viable cell concentrations in suspension culture from 100 cells/ml to 1 million cells/ml. The sensitivity of the method as a cytotoxicity assay increases if, following brief (1–4-h) exposure to drug, greater time is allowed for cell death and lysis to occur prior to flow cytometric counting of viable cells. If the viability assessment is deferred for at least 72 h following drug (daunorubicin, actinomycin D, vincristine) exposure, results were obtained approximating those obtained from the soft agar clonogenic assay or the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In studying the cytotoxic effects of vincristine, actinomycin D, 1-ß-D-arabinofuranosylcytosine, and daunorubicin on P388 or HL-60 cells sensitive and resistant to these agents, reasonable results were obtained by flow cytometric counting of viable cell number. We have been able to perform this flow cytometric viability assay with ease using bone marrow blast cells obtained from patients with acute myelogenous leukemia. The method is facile, relatively rapid, and since it is ideal for studying cells in suspension culture, its potential as a predictor of chemotherapeutic response in leukemia warrants further evaluation.

¹ Supported in part by Grant RO-1-CA40188 of NIH, National Cancer Institute (D. D. R.), and by the Medical Biotechnology Center of the Maryland Biotechnology Institute.

² Special Fellow of the Leukemia Society of America. To whom requests for reprints should be addressed, at the University of Maryland Cancer Center, Bressler Research Building, Room 9-015, 655 West Baltimore Street, Baltimore, MD 21201.

Received 12/23/87. Revised 1/18/89. Revised 4/11/89. Accepted 4/19/89.

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