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Journal of Lipid Research, Vol. 48, 961-967, April 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

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

A. J. Wensaas^*, A. C. Rustan, K. Lövstedt, B. Kull, S. Wikström^**, C. A. Drevon^* and S. Hallén^2,

^* Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway
Department of Pharmaceutical Biosciences, University of Oslo, Oslo, Norway
Department of Molecular Pharmacology, AstraZeneca R&D, S-431 83 Mölndal, Sweden
^** Department of Integrative Pharmacology, AstraZeneca R&D, S-431 83 Mölndal, Sweden

¹ An abstract of part of this study was presented at the 2006 International Society for the Study of Fatty acids and Lipids, 7^th Congress, Cairns, Australia, July 23–28, 2006.

Published, JLR Papers in Press, January 9, 2007.

² To whom correspondence should be addressed. e-mail: stefan.hallen{at}astrazeneca.com

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.

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

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