A cytotoxicity assay utilizing a fluorescent dye that determines accurate surviving fractions of cells

A cytotoxicity assay has been developed based on the measurement of the proliferative activity of surviving cells as quantified by a cell-incorporated fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The BCECF proliferative assay is fast (the results are obtained within 3-4 days depending on the cell line), accurate, not labor-intensive, does not require the use of radioisotopes or toxic compounds, and is amenable to automation. The BCECF proliferative assay was compared with two other indirect cytotoxicity tests, a trypan blue exclusion test and a BCECF viability test. Neither of these two latter assays reflected in any way the killing of cells by ricin. In contrast, using the BCECF proliferation assay, an optimal period of cell culturing after exposure to a toxin could be found so that the cytotoxicity values produced agreed with the surviving fractions of cells measured in a direct cytotoxicity assay. Under non-optimal conditions, the assay reflected the cell kill only qualitatively. Although it is common practice to conduct indirect cytotoxicity tests without validating them with a direct assay, our experiments demonstrate that the values obtained in such non-optimized indirect cytotoxicity tests may not parallel the cell kill and may, therefore, be meaningless.