1 14c palmitate

Rates of fatty acid oxidation were determined in primary brown adipocytes according to the conversion of [1–¹⁴C] palmitate (55 mCi/mmol: American Radiolabeled Chemicals; St. Louis, MO, USA) into ¹⁴C-acid soluble metabolites (ASM) and -CO₂[18] (link). Briefly, matured brown adipocytes were incubated for 6 h in 2 ml of fresh culture medium with 10% FBS supplemented with 1 mM carnitine (Sigma–Aldrich) and 0.25 μCi [1–¹⁴C] palmitate/ml plus 200 μM palmitate conjugated with fatty acid-free BSA. To determine ASM, the culture medium was mixed in conical tubes with 200 μl of 70% perchloric acid (Fisher Scientific; Pittsburgh, PA, USA) with filter papers soaked in 2N NaOH placed in the caps in order to trap ¹⁴C–CO₂. After incubation for 1 h on a horizontal shaker, the filter papers were removed, and the acidified medium was incubated overnight at 4 °C and then centrifuged at 14,000 × g for 20 min ¹⁴C-labeled CO₂ in the filter paper and ASM in the supernatant were dissolved in Ecoscint H (National Diagnostics; Atlanta, GA, USA) and were counted using a LS6000IC liquid scintillation counter (Beckman Coulter; Danvers, MA).