Analysis of Cell Viability by the MTT Assay

Among viability assays that depend on the conversion of substrate to chromogenic product by live cells, the MTT assay is still among one of the most versatile and popular assays. The MTT assay involves the conversion of the water-soluble yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to an insoluble purple formazan by the action of mitochondrial reductase. Formazan is then solubilized and the concentration determined by optical density at 570 nm. The result is a sensitive assay with excellent linearity up to ∼10⁶ cells per well. As with the alamarBlue assay, small changes in metabolic activity can generate large changes in MTT, allowing one to detect cell stress upon exposure to a toxic agent in the absence of direct cell death. The assay has been standardized for adherent or nonadherent cells grown in multiple wells. The protocol uses a standard 96-well plate. This can be scaled up, however, to suit a different plate format. Plate 500-10,000 cells per well in a 96-well plate. The assay has good linearity up to 10⁶ cells.