We perform MTT-Assays for compound screening in living cells. This assay monitors relative cell numbers in comparison to vehicle treated cells and provides a read out for an impact of a compound on the proliferation/cytotoxicity axis.
The priniciple of this assay is that the yellow-coloured water soluble salt 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) is reduced by mitochondrial Succinate-Dehydrogenases and the reduction equivalents NADH and NADPH to a violet-coloured water-insoluble Formazan salt. Since this reduction of Tetrazoliumbromid is an enzyme-dependent step, it can only occur in living cells (the more living cells, the more Tetrazoliumbromid conversion, the more violet color). The drawback of this assay is that we cannot make any conclusions whether a compound is cytostatic or cytotoxic. Nevertheless, the MTT-Assay is an inexpensive readout that requires only a limited handling time and that gives a good read-out about the biologic activity of a chemical compound and is therefore frequently the method of choice for screening of compounds.
To perform the assay, cells are incubated with MTT dissolved in cell culture medium (usually for 2-3 hours). Then the medium is aspirated and the water-insoluble Formazan salt is solubilized with 2-Propanol. The intensity of the violet color is determined with an ELISA reader at 550 nm.