Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture.
A new tetrazolium analog of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was evaluated as a substitute for MTT in the microculture screening assay for in vitro cell growth. This new tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), in the presence of phenazine methosulfate (PMS), gave a water-soluble formazan product that had an absorbance maximum at 490-500 nm in phosphate-buffered saline. The amount of colored product formed was proportional to the number of cells and the time of incubation of the cells with MTS/PMS. MTS/PMS was reactive in all the cell lines tested which included mouse leukemia L1210 cells, mouse Ehrlich tumor cells, mouse 3T3 fibroblasts, and human colon tumor cells (HT-29). HT-29 and 3T3 fibroblasts reduced MTS/PMS more efficiently than they reduced MTT. Comparable to the amount of product formed from MTT, MTS/PMS gave excellent product formation. The IC50 value for pyrazoloimidazole obtained using MTS/PMS was 200 microM; for 5-fluoro-2'-deoxyuridine, the IC50 value was 0.9 nM. These values compared very favorably with the IC50 values obtained by direct cell counts. Further, the same IC50 values were obtained when the absorbances of the formazan product in the 96-well plates were determined after different times of incubation.
Cory
A H
AH
Department of Biochemistry, East Carolina University School of Medicine, Greenville, NC 27858.
Owen
T C
TC
Barltrop
J A
JA
Cory
J G
JG
D000818:Animals; D002452:Cell Count; D002455:Cell Division; D002478:Cells, Cultured; D004396:Coloring Agents; D004341:Drug Evaluation; D004354:Drug Screening Assays, Antitumor; D005562:Formazans; D006801:Humans; D008773:Methylphenazonium Methosulfate; D051379:Mice; D013053:Spectrophotometry; D013778:Tetrazolium Salts / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry; D014407:Tumor Cells, Cultured
D004396:Coloring Agents; D005562:Formazans; D013778:Tetrazolium Salts; D013844:Thiazoles; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; D008773:Methylphenazonium Methosulfate; C022616:thiazolyl blue