Mechanistic deconvolution of autoreduction in tetrazolium-based cell viability assays

Optical reporters for biochemical activities within cells are a powerful way to quantify the physiological responses of cellular systems to experimental conditions. For example, tetrazolium dyes, such as 5-diphenyl tetrazolium bromide (MTT), can be used to quantify the number of living cells present in a sample. This is useful for the determination of the toxic or antiproliferative activity of certain conditions that a cell might be exposed to, such as the administration of anticancer compounds. Inside of an active, living cell, tetrazolium salts, such as MTT, are reduced to brightly-colored formazan dye counterparts, which can be measured spectrophotometrically as a proxy for cellular viability. However, it has been reported that small molecules may interfere with the results of an MTT assay, at times impacting the accuracy or reproducibility of the results of the experiment. In this study, to examine if reducing agents were a source of inaccuracy in the MTT assay, we tested common reducing agents in cellular media, which are commonly used in biological assays, for unforeseen autoreduction and determined that ascorbic acid, cysteine, and glutathione (GSH) were reactive with MTT under basic conditions. We also found pH accelerated autoreduction, with a pH of 10 correlating with the most autoreduction. We advise to use media specially depleted of ascorbic acid, cysteine, and GSH, or a higher cellular density for such assays, in order to prevent interference through autoreduction.