Abstract

A reliable, indirect method (GPD/INT assay) for estimating the number of live animal cells in multiwell culture has been devised. It is based on the glucose-6-phosphate dehydrogenase (Gpdh) and 6-phosphogluconate dehydrogenase activities present in the cytoplasm of viable eukaryotic cells but not in their bathing medium nor in nonviable cells. A single reagent mixture, buffered at pH 7.8 and containing Tris, Triton X-100, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate, and iodonitrotetrazolium violet, is added to the cultures. The Triton X-100 releases the cytoplasmic contents into the medium, facilitating enzyme-catalyzed oxidation of the glucose-6-phosphate and 6-phosphogluconate by NADP. The resulting reduced nicotinamide adenine dinucleotide phosphate, NADPH, reduces tetrazolium violet to its formazan, the color of which reflects the number of living cells that were in the culture. The assay was tested on recombinant Gpdh and the several types of animal and insect cell lines to verify the premise that there is proportionality between the amount of GPdh and number of viable cells in the cultures. The method has been used to quantitate the effects of growth inhibitors on cells in 96-well cultures.