Measuring Fatty Acid Oxidation in Thymic Lymphocytes

Fatty acid oxidation was measured using ³H-palmitate as previously described (19 (link)). Briefly, 2 × 10⁶ freshly isolated thymic lymphocytes were resuspended in 1 mL of modified KHB medium (111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl₂, 2 mM MgSO₄, 1.2 mM NaH₂PO₄, 2.5 mM glucose, 0.5 mM carnitine, 5 mM HEPES, pH 7.4) with or without 200 μM etomoxir. A mixture of [9,10-³H] palmitate (53.7 Ci/mmol, PerkinElmer, Boston, MA) and 10% BSA in KHB buffer was added to the cell suspension and incubated at 37 °C for 2.5 h. After incubation, the cells were spun down at 300x g and assayed for protein content while the supernatant was subjected to chloroform-methanol extraction to measure the ³H₂O product in the aqueous phase. The etomoxir-sensitive ³H₂O generated was used to calculate the β-oxidation rate and expressed as CPM/μg protein.