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 follow
ing 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 two different established tumor cell lines. Results were
based upon comparisons of the ratios of "viable" tumor cells
to duck erythrocytes on permanent cytocentrifuge slides pre
pared 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 three assays demonstrated a clear
dose-effect relationship for most of the drugs tested. Dye
exclusion assays may have a useful role in chemosensitivity
testing in vitro.