The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay is widely used for in vitro measurement of the metabolic viability of cell cultures subjected to different culture conditions. This convenient assay, which is based on the ability of viable cells to produce formazan, can be affected significantly by a number of conditions. These conditions can be roughly divided into groups, firstly influences which affect the spectrum of the produced formazan and secondly influences which affect the amount of formazan produced per cell. We studied the various chemical and biochemical aspects involved in the MTT assay. Our data indicated that microscopical viewing of cell cultures before and after performing the assay, a medium renewal with a well-defined MTT-incubation medium at the end of the culture period and regular cell counting are essential steps to ensure a reliable performance of the MTT assay. In conclusion, providing the necessary precautions are taken, the MTT assay can be used reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival.