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Concept
MTT assay
Summary
The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT, which is chemically 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes including XTT, MTS and the WSTs, are used in conjunction with the intermediate electron acceptor, 1-methoxy phenazine methosulfate (PMS). With WST-1, which is cell-impermeable, reduction occurs outside the cell via plasma membrane electron transport. However, this traditionally assumed explanation is currently contended as proof has also been found of MTT reduction to formazan in lipidic cellular structures without apparent involvement of oxidoreductases.
Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light.
MTT, a yellow tetrazole, is reduced to purple formazan in living cells. A solubilization solution (usually either dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. The degree of light absorption is dependent on the degree of formazan concentration accumulated inside the cell and on the cell surface. The greater the formazan concentration, the deeper the purple colour and thus the higher the absorbance.
XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) has been proposed to replace MTT, yielding higher sensitivity and a higher dynamic range.
Official source
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