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.
Plumb
J A
JA
C.R.C. Department of Medical Oncology, University of Glasgow, United Kingdom.
Milroy
R
R
Kaye
S B
SB
D001391:Azo Compounds / Q000493:pharmacokinetics*; D002021:Buffers; D002452:Cell Count; D004396:Coloring Agents / Q000493:pharmacokinetics*; D003470:Culture Media / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D005562:Formazans / Q000493:pharmacokinetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D010084:Oxidation-Reduction; D013057:Spectrum Analysis; D013778:Tetrazolium Salts / Q000493:pharmacokinetics*; D013844:Thiazoles / Q000493:pharmacokinetics*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014750:Vincristine / Q000494:pharmacology
D001391:Azo Compounds; D002021:Buffers; D004396:Coloring Agents; D003470:Culture Media; D005562:Formazans; D013778:Tetrazolium Salts; D013844:Thiazoles; D014750:Vincristine; D004317:Doxorubicin; C022616:thiazolyl blue