Cell-based multiwell assays for the detection of substrate accumulation and oxidation1

Journal of Lipid Research

S. Wikström¹  K. Lövstedt²  B. Kull²  S. Hallén²  A.J. Wensaas³  C.A. Drevon³  A.C. Rustan⁴ 

[1]Department of Integrative Pharmacology, AstraZeneca R&D, S-431 83 Mölndal, Sweden

[2]Department of Molecular Pharmacology, AstraZeneca R&D, S-431 83 Mölndal, Sweden

[3]Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway

[4]Department of Pharmaceutical Biosciences, University of Oslo, Oslo, Norway

关键词: fatty acids;    glucose;    uptake;    CO2 capture;    scintillation proximity assay;   

DOI  :  

来源: DOAJ

【 摘 要 】

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. 14CO2 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.

【 授权许可】

Unknown