The cells were kept at 37°C and 5% CO2. To test the individual and combined effects of glucose depletion and fatty acid stimulation on the free CoA level and acyl-CoA profile, the cells were incubated for 24 hours in two different conditions. In condition 1 the cells were cultured in DMEM (5 mM glucose, 1 mM pyruvate supplemented with 3 mM glutamine and 10% FBS) with additional supplements 2 mM L-carnitine and 0.5 mM BSA-bound palmitate. Condition 2 was with glucose-free DMEM (no glucose, no glutamine, no pyruvate, 10% FBS) supplemented with 2 mM L-carnitine and 0.5 mM BSA-bound palmitate. After 24 hours, the cells were washed twice with ice-cold PBS and harvested for further analysis. Condition 1 cells were "supplemented cells" with multiple carbon sources, while condition 2 cells were "starved cells" with fewer carbon sources.