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 Assay1

The tetrazolium dye, 3-(4,5-dimethylthia/ol-2-yl)-2,5-diphi-iiyltclrazolium 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 M l'I forma/an production. We have shown that reduction of MTT to M l'I forma/an by cells is dependent on the amount of MTT in the incubation medium. The concentration required to give maximal MTT-formazan productiondiffers 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 productionand, hence, cell number at high cell densities. This error can result in a 10-fold underestimation of chemosensitivity. Addition of a bufferat pH 10.5 to the solubili/ed MTT-formazan productcan overcome the effects of both cell density and culture medium on the absorption spectrum. Provided that sufficient MIT is used and the pH of the MTTformazan productis controlled, dye reductioncan be used to estimate cell numbers in a simple chemosensitivity assay the results of which agree well with a commonly used clonogenic assay.