Cell-based multiwell assays for the detection of substrate accumulation and oxidation.
We describe multiwell assays for detecting the accumulation as well as the subsequent oxidation of (14)C-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 (14)CO(2) 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. (14)CO(2) is captured in the filter and quantified by conventional scintillation. We demonstrate both the accumulation and subsequent oxidation of (14)C-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.
Wensaas
A J
AJ
Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway.
Rustan
A C
AC
Lövstedt
K
K
Kull
B
B
Wikström
S
S
Drevon
C A
CA
Hallén
S
S
D017667:Adipocytes / Q000378:metabolism; D002245:Carbon Dioxide / Q000032:analysis*; D002250:Carbon Radioisotopes / Q000032:analysis; D002478:Cells, Cultured; D019411:Clinical Laboratory Techniques; D004867:Equipment Design; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008660:Metabolism; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D012588:Scintillation Counting / Q000379:methods / Q000592:standards
D002250:Carbon Radioisotopes; D002245:Carbon Dioxide