COMPARISON OF DYE EXCLUSION ASSAYS WITH A CLONOGENIC-ASSAY IN THE DETERMINATION OF DRUG-INDUCED CYTO-TOXICITY

Abstract

The following factors must be considered when dye exclusion assays are interpreted. (a) It may require several days for lethally damaged cells to lose their membrane integrity following a cytotoxic insult. (b) During this time, the surviving cells may continue to proliferate. (c) Also during this time, some lethally damaged cells may undergo an early disintegration, so that they are not present to be stained with dye at the end of the culture period. Factors b and c may cause an underestimate of cell kill when the results of the assay are based upon the traditional percent viability expression. In order to overcome these problems, an internal standard was developed and tested. This was based upon the addition of a constant number of permanently fixed duck erythrocytes to the cultures of cells from 2 different established tumor cell lines [HL-60 human promyelocytic leukemia cells, mouse tumor MDA4-D2 cells]. Results were based upon comparisons of the ratios of viable tumor cells to duck erythrocytes on permanent cytocentrifuge slides prepared from the cultures. This novel ratio method was found to be a more sensitive index of drug-induced cell kill than the traditional percent viability method. A standard agar cloning assay gave somewhat higher estimates of cell kill than the ratio method, although both assays were in qualitative agreement for the drugs tested. All 3 assays demonstrated a clear dose-effect relationship for most of the drugs tested [5-fluorouracil, nitrogen mustard, methotrexate, vinblastine, doxorubicin, actinomycin D, 1,3-bis(2-chloroethyl)-1-nitrosourea, cis-diamminedichloroplatinum]. Dye exclusion assays may have a useful role in chemosensitivity testing in vitro.