Optimization of the Tetrazolium Dye (MTT) Colorimetric Assay for Cellular Growth and Viability

The MTT colorimetric assay is an established method of determining viable cell number in proliferation and cytotoxicity studies. This assay is based on the cleavage of the yellow tetrazolium salt, MTT, to form a soluble blue formazan product by mitochondrial enzymes, and the amount of formazan produced is directly proportional to the number of living, not dead cells, present during MTT exposure. Since the MTT assay is rapid, convenient, and economical, it has become a very popular technique for quantification of viable cells in culture. However, various parameters have been identified that can affect cellular metabolism and other factors, which significantly modify MTT-specific activity and can result in calculated false high or false low cell counts. Therefore, it is essential to establish assay parameters with the proper controls for each cell line and/or drug treatment in order to optimize assay conditions and minimize confounding effects. These parameters should include determining appropriate cell densities, culture medium, optimal concentrations and exposure times for MTT, fresh culture medium at the time of assay to avoid nutrient depletion, and controlling for drug treatment effects that may influence cellular metabolism. By controlling these important parameters, the MTT colorimetric assay provides accurate and reliable quantification of viable cell number.