MTT assay.html

The MTT assay and the MTS assay are laboratory tests and standard colorimetric assays (an assay which measures changes in color) for measuring the activity of enzymes that reduce MTT or MTS + PMS to formazan, giving a purple color. This mostly happens in mitochondria, and so the assays are therefore largely a measure of mitochondrial activity. It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials, since those agents would result in cell toxicity and therefore mitochondrial dysfunction and therefore decreased performance in the assay.

Yellow MTT (3-(4,5-Di methyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in the mitochondria of living cells.¹ 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 absorption maximum is dependent on the solvent employed.

MTS is a more recent alternative to MTT. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (PMS), produces a water-soluble formazan product that had an absorbance maximum at 490-500 nm in phosphate-buffered saline.² It is advantageous over MTT in that (1) the reagents MTS + PMS are reduced more efficiently than MTT, and (2) the product is water soluble, decreasing toxicity to cells seen with an insoluble product.

These reductions take place only when mitochondrial reductase enzymes are active, and therefore conversion is often used as a measure of viable (living) cells. However, it is important to keep in mind that other viability tests sometimes give completely different results, as many different conditions can increase or decrease metabolic activity. Changes in metabolic activity can give large changes in MTT or MTS results while the number of viable cells is constant. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death, or changing metabolism of cells, can be deduced through the production of a dose-response curve.

References

^ Mosmann T (December 1983). "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays". Journal of immunological methods 65 (1-2): 55–63. PMID 6606682, http://linkinghub.elsevier.com/retrieve/pii/0022-1759(83)90303-4.
^ Cory AH, Owen TC, Barltrop JA, Cory JG (July 1991). "Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture". Cancer communications 3 (7): 207–12. PMID 1867954.

Wilson, A. P., Cytotoxicity and Viability Assays in Animal Cell Culture: A Practical Approach, 3rd ed. (ed. Masters, J. R. W.) Oxford University Press: oXford 2000, Vol. 1,

MTT assay.html

References

See also