Isotopic Labeling and Metabolomic Analysis

NRCMs were incubated in 6-well plates for 5 min or 4–18 h in DMEM media (US Biological; Swampscott, MA, USA) containing 1 mM pyruvate, 4 mM glutamine, and 25 mM [¹³C₆]-glucose (99% purity, microbiological and pyrogen tested; Cambridge Isotope Laboratories, Inc., Tewksbury, MA). Additionally, for compound/inhibitor experiments, FCCP, KA, Oligo, Rot, or 2DG were added to this [¹³C₆]-glucose containing media. After the specified isotope labeling time point, cell reactions were quenched in cold acetonitrile, and extracted in acetonitrile:water:chloroform (v/v/v, 2:1.5:1), as described previously^{31 (link),32 (link),60 (link),61 (link)}, to obtain the polar, nonpolar, and insoluble proteinaceous fractions. The nonpolar (lipid) layer was collected, dried under a stream of nitrogen gas, and reconstituted in 0.1 ml of chloroform:methanol:butylated hydroxytoluene (2:1 + 1 mM) mixture and stored at −80 °C for future analysis. The polar fraction was lyophilized using a Freezone 2.5 L −84 °C benchtop freeze dryer (Labconco, Kansas City, CO, USA). The dried sample was reconstituted in 100 μl 20% acetonitrile and used for LC-MS analysis.