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

• Published in: Journal of lipid research Vol. 48; no. 4; pp. 961 - 967
• Main Authors: Wensaas, A.J., Rustan, A.C., Lövstedt, K., Kull, B., Wikström, S., Drevon, C.A., Hallén, S.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01-04-2007; Elsevier
• Subjects: CO2 capture; fatty acids; glucose; scintillation proximity assay; uptake; glucose; CO2 capture; fatty acids; uptake; scintillation proximity assay
• ISSN: 0022-2275; 1539-7262
• DOI: 10.1194/jlr.D600047-JLR200

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.