Cell-based multiwell assays for the detection of substrate accumulation and oxidation

Abstract

We describe multiwell assays for detecting the accumulation as well as the subsequent oxidation of 14C-labeled substrates in cultured cells. Accumulation is monitored in real time by an established scintillation proximity assay in which the scintillator is embedded in the plate base primarily detecting cell-associated radiolabel. The substrate oxidation assay is a novel variant of previously described experimental approaches aimed at trapping 14CO2 produced by isolated enzymes, organelles, or intact cells. This method uses a standard 96-well tissue culture plate and, on top, an inverted filter plate immersed with NaOH that are clamped into a sandwich sealed with a silicon gasket to obtain gas-tight compartments. 14CO 2 is captured in the filter and quantified by conventional scintillation. We demonstrate both the accumulation and subsequent oxidation of 14C-labeled substrates in cultured human myotubes, adipocytes, and hepatocytes. Both methods are adaptable for compound screening; at the same time, these protocols provide easy-to-use and time- saving methods for in vitro studies of cellular fuel handling. Copyright ©2007 by the American Society for Biochemistry and Molecular Biology, Inc.