出版社：American Society for Biochemistry and Molecular Biology
摘要：We describe multiwell assays for detecting the accumulationas well as the subsequent oxidation of 14C-labeled substratesin cultured cells. Accumulation is monitored in real time byan established scintillation proximity assay in which the scintillatoris embedded in the plate base primarily detecting cell-associatedradiolabel. The substrate oxidation assay is a novel variantof previously described experimental approaches aimed at trapping14CO2 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 areclamped into a sandwich sealed with a silicon gasket to obtaingas-tight compartments. 14CO2 is captured in the filter andquantified by conventional scintillation. We demonstrate boththe accumulation and subsequent oxidation of 14C-labeled substratesin cultured human myotubes, adipocytes, and hepatocytes. Bothmethods are adaptable for compound screening; at the same time,these protocols provide easy-to-use and time- saving methodsfor in vitro studies of cellular fuel handling.Supplementary key words fatty acids • glucose • uptake • CO2 capture • scintillation proximity assay
Abbreviations: CPT1, carnitine palmitoyl transferase-1; DOG, deoxyglucose; DPBS, Dulbecco's phosphate-buffered saline; EPA, eicosapentaenoic acid; FCS, fetal calf serum; IBMX, 3-isobutyl-1-methylxanthine; LA, linoleic acid; OA, oleic acid; PA, palmitic acid; P/S, penicillin/streptomycin; SGBS, Simpson-Golabi Behmel syndrome; SPA, scintillation proximity assay
