The
hydrogen acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium
bromide (MTT) is commonly utilized to estimate cellular viability in
drug screening protocols. The present investigation was prompted, in part, by observations that reduction of MTT to its colored reaction product,
MTT formazan, varied between cell lines and with culture age. A correlation was established between the
D-glucose concentration of the culture medium at the time of assay and the production of
MTT formazan for cell lines representing seven
tumor histologies. A decrease in the concentration of
D-glucose from culture medium was accompanied by a decrease in MTT specific activity (
MTT formazan/microgram cell
protein) for a number of cell lines. Cells which extensively metabolized
D-glucose exhibited the greatest reduction in MTT specific activity. Further evidence that the
D-glucose concentration of the culture medium played an important role in MTT reduction was provided by experiments which demonstrated that transfer of cells to a
glucose-free medium (L-15) was accompanied by an immediate decrease in MTT reduction which was pH independent. These studies suggested that cellular transport and constant metabolism of
glucose were required for maximum MTT reduction. Decreases in the cellular concentration of the reduced
pyridine nucleotides NADH and
NADPH were accompanied by concomitant decreases in
MTT formazan production.
MTT formazan varied significantly among cell lines in both the kinetics of its formation and the degree of saturability exhibited. Apparent IC50 values for
Adriamycin varied, in a cell line-specific manner, with MTT exposure time. These results indicate that MTT specific activity is significantly influenced by a number of parameters and suggest that assay conditions should be established which minimize their effects.