Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities

A method is described for estimating the numbers of animal cells in multi-w ell culture by simultaneously measuring the lactate dehydrogenase activity of the total culture and the medium. The difference between the two reflect s the dehydrogenase content of the cells and correlates with cell number. T his LDH/INT method was tested using several lines of normal and transformed suspension and adherent cells. The lactate dehydrogenase activities of dup licate cultures were determined colourimetrically using reaction cocktails containing lactate, NAD(+), diaphorase, and p-iodonitrotetrazolium violet, with and without Triton X-100. The difference in absorbance at 490 nm (Delt a A(490) = A(490), test - A(490, control)) was used to calculate the lactat e dehydrogenase activity of the total culture (+ Triton) and the medium (- Triton). The cellular lactate dehydrogenase activity (difference between to tal and medium dehydrogenase activities) was proportional to viable cell nu mber. The effects on cell growth of four metabolic inhibitors, sodium azide , actinomycin D, cycloheximide, and taxol, were determined using the LDH/IN T assay and direct cell counting. The inhibitor concentrations that caused decreases in the LDH activity and cell number by 50% were similar. The LDH/ INT assay is quick and sensitive, works equally well for adherent and suspe nsion cells, and provides information about LDH activities of both the medi um and cells. It is particularly useful for screening potential cell-growth inhibitors.