2 deoxy d glucose

Cells were seeded in Seahorse 96-well plates and differentiated for eight days with 1.8 mM calcium (control), high magnesium (10 mM), high calcium (10 mM) or calcium free conditions for 2–8 days. The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured with a XF96 Extracellular Flux analyzer (Seahorse Bioscience, Agilent technologies). Cells were equilibrated at 37 °C in XF Assay Medium Modified DMEM (Seahorse Bioscience) supplemented with 25 mM glucose one hour prior measurement. All compounds were diluted as ten-fold concentrations in assay medium and loaded in the equilibrated cartridge ports as follows: A) 20 µg/ml oligomycin (Merck), B) 10 µM FCCP (R&D systems) +/− 50 mM sodium pyruvate (Gibco), C) 25 µM rotenone and antimycin A (Sigma), D) 1 M 2-deoxy-D-glucose (Alfa Aesar). Each cycle comprised two minutes each of mixing, waiting and measuring. Non-mitochondrial respiration (OCR) and non-glycolytic acidification (ECAR) were subtracted from other values to determine mitochondrial oxygen uptake. For the analysis, the mean values of either the last two cycles prior injection (basal respiration, acidification/glycolysis) or the first two after injection (sum of ATP production and proton leak or maximal respiration (OCR) and maximal acidification/glycolytic capacity (ECAR)) were averaged and calculated.

Primary brown preadipocytes were cultured and differentiated on XF24 Seahorse plates (Agilent technologies Inc., Santa Clara, CA, USA). For each biological replicate, brown preadipocytes from two mice were pooled. At day 7 of differentiation, brown adipocytes were pre-treated with 400 µM L-serine for 3 h. Prior to measurement and following the pre-treatment, DMEM was changed to XF Assay Medium-Modified DMEM Agilent technologies Inc., Santa Clara, CA, USA) supplemented with or without 400 µM L-serine. Assay ports were loaded with: (A) assay media, 4 mM L-serine, 10 µM isoproterenol or 4 mM L-serine and 10 µM isoproterenol, (B) 20 µg/mL oligomycin (Merck, Darmstadt, Germany), (C) 20 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (R&D systems, Wiesbaden, Germany), (D) 25 µM rotenone; 25 µM antimycin A (Sigma Aldrich, Taufkirchen, Germany) and 1 M 2-deoxy-D-glucose (Alfa Aesar, Kandel, Germany). Oxygen consumption rates (OCR) were measured at day 7 of differentiation with the Seahorse XF24 Analyzer (Seahorse Bioscience-Agilent, North Billerica, MA, USA). Each cycle consisted of 2 min mixing, 2 min waiting and 2 min measuring. Data analysed with Wave software version 2.6.1.53 (Agilent technologies Inc., Santa Clara, CA, USA).