Antioxidant compounds interfere with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide cytotoxicity assay

Antioxidants are often added to culture media as cytoprotective agents. We examined the effects of antioxidants on the results and interpretation of t he 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability. Without cells, the thiol-containing antioxidant compou nds B-mercaptoethanol, dithiothreitol, pyrrolidine-dithiocarbamate, and N-a cetyl-L-cysteine (acetylcysteine) reduced MTT tetrazolium salts to a blue f ormazan product in a dose-dependent manner. Addition of the compounds L-asc orbic acid and (+)-alpha -tocopherol acid succinate had different effects. In contrast, addition of the antioxidants N-acetyl-5-methoxytryptamine and (-)-2-oxo-4-thiazolidine carboxylic acid, which do not contain reactive thi ol groups, did not result in the development of blue formazan product. Thes e results showed that antioxidants, and potentially other chemotherapeutic compounds that contain free thiol groups or other reducing equivalents, rea dily reduce MTT to produce the blue formazan, irrespective of the viability of the cells present. This undescribed reaction can, therefore, significan tly influence the results and interpretation of cell-viability experiments.