96-well plate assay for sublethal metabolic activity.

A 96-well plate assay has been devised for estimation of sublethal metabolic activity for compounds administered to in vitro cell cultures during 6- and 24-h exposures. This screen combines a resazurin reduction assay with lactate production and glucose consumption rate assays to assess effects of compounds on both culture viability and metabolic inhibition. In this article, the assay is demonstrated by determining the extent to which five glycolysis inhibitors, namely, phloretin, 2-deoxyglucose, iodoacetate, fluoride, and oxamate, induce metabolic inhibition without cell death for in vitro fibroblast cell cultures. During 6-h exposures, iodoacetate was found to be the most potent inhibitor of glycolysis, whereas iodoacetate and phloretin were most cytolethal. 2-Deoxyglucose had the largest sublethal metabolic range, spanning just over two orders of magnitude in concentrations between its IC(50) values for cytolethality and metabolic inhibition. This method provides a simple and inexpensive way to determine global metabolic and cytolethal effects of compounds for in vitro cell culture systems. It should be of use directly for large-scale screening and ranking of compounds during drug discovery and development, in conjunction with or following some more direct measure of therapeutic efficacy of a prospective drug candidate. Moreover, firsthand determination of the concentrations over which a compound has lethal, sublethal metabolic, or no such effects should serve as a cost-effective and time-saving first step in a given research study, preceding more expensive, lengthy, and/or detailed in vitro methods.