Determination of Cellular Fatty Acid Oxidation

The cellular FAO was determined following the published literature^{89 (link)} Briefly, 3 × 10⁵ cells were seeded into each well of a 12-well plate. After 12 h incubation, [9,10-³H(N)]-palmitic acid [0.5 µCi (~9.3 pmol)] (PerkinElmer, Catalog# NET043001MC) in 15 µl carrier solution (1 mM sodium palmitate, 0.17 mM BSA, 150 mM NaCl) was added to each well and incubated for 6 h. The [9,10-³H(N)]-palmitic acid was converted to ³H₂O via mitochondria FAO. To control the background, each experiment contained cell-free control. After labeling, 0.5 ml out of 1 ml culture medium was collected into a 15 ml tube with 100 µl of 1.2 N HCl, and an uncapped 0.5 ml tube containing 0.25 ml of sterile distilled water was carefully inserted into the tube and further incubated for 3 days, and the radioactivity present in the water and medium were determined by scintillation counting. The FAO rate of each sample was calculated and normalized to protein concentration.