Fluorescence-based viability assay for studies of reactive drug intermediates

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Abstract

Studies of drug toxicity, toxicologic structure-function relationships, screening of idiosyncratic drug reactions, and a variety of cytotoxic events and cellular functions in immunology and cell biology require the sensitive and rapid processing of often large numbers of cell samples. This report describes the development of a high-sensitivity, high-throughput viability assay based on (a) the carboxyfluorescein derivative 2′–7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) as a vital dye, (b) instrumentation capable of processing multiple small (<100 cells) samples, and (c) a 96-well unidirectional vacuum filtration plate. Double staining of cultured peripheral blood mononuclear cells with BCECF and propidium iodide (PI) showed no overlap between PI+ (nonviable) and BCECF+ (viable) cells by flow cytometric analysis. Optimal conditions were developed for dye loading and minimizing physical cell damage and fluorescence quench during the assay procedure. The ratio of BCECF fluorescence to internal standard fluorescent particles was linear from 40 to >20,000 cells with a signal:noise ratio of ∼3 at 40 cells/well. Sulfamethoxazole hydroxylamine (SMX-HA) was used as a model toxic drug metabolite to explore the validity of the BCECF procedure. SMX-HA, but not its parent compound sulfamethoxazole, resulted in a dose dependent loss of cellular fluorescence and the parallel accumulation of PI+ nonviable cells. When compared to the currently used tetrazolium dye reduction viability assay, the BCECF method was 3-fold more sensitive, >10-fold faster, and required 1101100 the cell numbers.

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This work was Supported by grants to HMD and SPS from the Medical Research Council, the National Cancer Institute, and the Muscular Dystrophy Foundation of Canada.

2

JSL is supported by a fellowship from the Medical Research Council of Canada.

3

SPS is a Scholar of the Medical Research Coucil of Canada.

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