USE OF AN AQUEOUS SOLUBLE TETRAZOLIUM FORMAZAN ASSAY TO MEASURE VIABILITY AND PROLIFERATION OF LYMPHOKINE-DEPENDENT CELL-LINES

A new tetrazolium compound, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt), has recently been described which in the presence of phenazine methosulfate (PMS) is reduced by living cells to yield a formazan product that can be assayed colorimetrically. An important advantage of MTS/PMS over other tetrazolium dyes (e.g., MTT) is the aqueous solubility of the reduced formazan product which eliminates the need for detergent solubilization or organic solvent extraction steps. Its advantages over XTT/PMS, another tetrazolium which yields a water-soluble formazan product, include the absorbance range of color produced (515-580 nm as opposed to 450 nm), the rapidity of color development, and the storage stability of the MTS/PMS reagent solution. In the present study, MTS/PMS was used to assay viability and proliferation of the IL-2-dependent HT-2 and CTLL-2 cell lines and the IL-3-dependent FDC-PI and FL5.12 cell lines. With each cell line, the amount of formazan product was time-dependent and proportional to the number of viable cells. Furthermore, with both HT-2 and CTLL-2 cells it was found that cultures could be simultaneously labeled with MTS/PMS and [H-3]thymidine, with relatively little effect of the dye on uptake of the latter. This feature was further capitalized upon in studies with FDC-P1 cells, in which the co-addition of MTS/PMS and [H-3]thymidine was used to distinguish between cell viability and proliferation.