Limitations of the MTT Assay in Cell Viability Testing

Background. The MTT assay is widely recommended for examining the cytotoxic effect of xenobiotics, assessing proliferation rates, and analyzing cell activity. The aim of this study was to evaluate the usefulness of the MTT assay in examining human lymphocyte viability in own cell culture systems which contained fluphenazine (FPh), a suspected cancer chemopreventive agent, and doxorubicin (DOX), an anticancer drug, in cell culture medium. Material and Methods. Cell culture viability was estimated with two tests: the MTT assay and the propidium iodide (PI) exclusion assay. These results were compared using the standard ANOVA procedure. Results. The propidium iodide exclusion−test, a microscopic assay of cell culture viability, revealed an increase in dead cell number in cultures treated with DOX and an absence of viable cell changes in cultures treated with FPh in the range of the tested concentrations. The results obtained with the MTT reduction assay in the cultures gave the opposite results; the assay did not mirror the real viable cell number in these cultures. Statistical analysis demonstrated that the results obtained with the two assays differed significantly. Conclusions. This study shows that the MTT assay is not a feasible tool for determining cell viability in cultures in which DOX and FPh are used and possible sources of the limitations of the MTT assay are discussed (Adv Clin Exp Med 2008, 17, 5, 525–529).