An improved method has been developed to count cells in situ based on the measurement of esterase activity with carboxyfluorescein diacetate. This sensitive, semiautomated microplate fluorometer assay was able to estimate viable cell numbers over a range of 5 x 10(2) to 2.6 x 10(5) cells/well in a tumor cell line. Sensitivity to 10(3) was demonstrated in two other cell lines. Sub- and supranormal fluorescence events which can be responsible for unreliable readings when using a fluorescence assay for cell counting were quantified in a menadione (cytotoxic agent)/U-87 MG (cell line) model. There was a close correlation between the fluorometer method and Coulter counter method for two different tumor cell lines when this method was performed on cells after sub- and supranormal fluorescence events had ceased.
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