BioProtocols--C2

MTT Assay

Background of MTT Assay: MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, first described by Mosmann in 1983, is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form a dark blue formazan crystals which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. Solubilisation of the cells by the addition of a detergent results in the liberation of the crystals which are solubilized . The number of surviving cells is directly proportional to the level of the formazan product created. The color can then be quantified using a simple colorimetric assay. The results can be read on a microreader under a specific wavelength.

Reference
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55-63.

A Mini Protocol for MTT Assay in a 24 Well Format Plate

-Plate cells.

-Remove media.

-Wash w/ 1x500 ul 1x PBS.

-Replaced w/ 200 ul medium plus 10 ul 5 mg/ml MTT stock.

-Incubate 2h at 37 degree centigrade

-Remove the MTT mixture.

-Replace w/ 200 ul DMSO.

-Transferred the DMSO solution to 96 format plate well, respectively.

-Read the plate with a microreader at wavelength 550 nm-650 nm.

MTT Cell Assay Protocol

MTT Viability Assay