Fatty acid oxidation (β-oxidation) assay

Fatty acid oxidation was measured in triplicate by quantitating the production of ³H₂O from [9,10-³H]palmitate as described previously⁵⁸. Briefly, 200 µl per well of PBS containing 1 µCi [9,10-³H] palmitate (30 Ci/mmol, PerkinElmer Life Science Products) bound to fatty acid-free albumin was added to MIAPaCa-2 cells in 24-well microplates. Incubation was carried out for 2 h at 37 °C. In some experiments, 25 µM etomoxir or 500 µM l-carnitine was added to the incubation medium. After incubation, the mixture was removed and added to 200 µl of cold 10% trichloroacetic acid. The tubes were centrifuged for 10 min at 2200×g at 4 °C, and aliquots of supernatants (350 µl) were removed, mixed with 55 µl of 6 N NaOH, and applied to ion-exchange resin (Dowex 1×2-400 resin, Sigma-Aldrich). The columns were washed twice with 750 µl of water, and the eluates were counted. Palmitate oxidation rates were expressed as ³H₂O counts/10⁶ cells/h.