Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay.

The tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), is reduced by live but not dead cells, and this reaction is used as the end point in a rapid drug-screening assay. It can also be used for accurate determinations of drug sensitivity but only if a quantitative relationship is established between cell number and MTT-formazan production. We have shown that reduction of MTT to MTT-formazan by cells is dependent on the amount of MTT in the incubation medium. The concentration required to give maximal MTT-formazan production differs widely between cell lines. The absorption spectrum of MTT-formazan varies with cell number and with pH. At a low cell density or a high pH, the absorption maximum is at a wavelength of 560 to 570 nm. However, at a high cell density or a low pH, there are two absorption maxima; one at 510 nm and a second at about 570 nm. Measurements of absorbance at 570 nm underestimate MTT-formazan production and, hence, cell number at high cell densities. This error can result in a 10-fold underestimation of chemosensitivity. Addition of a buffer at pH 10.5 to the solubilized MTT-formazan product can overcome the effects of both cell density and culture medium on the absorption spectrum. Provided that sufficient MTT is used and the pH of the MTT-formazan product is controlled, dye reduction can be used to estimate cell numbers in a simple chemosensitivity assay the results of which agree well with a commonly used clonogenic assay.