Measuring Fatty Acid β-Oxidation in Cells

β-oxidation of fatty acids was measured as previously described^{65 (link)}. Briefly, we plated 30 x 10⁴ cells in 6-well plates and 18h later, we incubated the cells with medium supplemented with [9, 10-³H]-palmitate (Hartmann Analytic GmbH, 1 mCi/mL) bound to 10% fatty acid free bovine serum albumin (Sigma). A total of 3.3 μl of [9, 10-³H]-palmitate and 6.7 μl of fatty acid free bovine serum albumin were used per 1 ml of cell culture medium and cultured for 24 h. After 24 h, supernatant was applied to ion-exchange columns (Dowex 1X8–200, Sigma), and tritiated water was recovered by eluting with 0.5 ml of H₂O. A 100μl aliquot was then mixed with 100μl of liquid scintillation cocktail and measured the amount of radioactivity released with a Top count NXT liquid scintillation counter (Packard) in 96-well polystyrene plates (Nunc F96 microwell, Thermo). β-oxidation was expressed as counts per minute (CPM) per milligram of protein.