Palmitate β-Oxidation Assay in CD4+ T Cells

The isolated human CD4+ cells were cultured at 4 × 10⁵ cells/well in a 48-well plate in RPMI and were activated with anti-CD3/anti-CD28-coated beads. The palmitate β-oxidation was evaluated as previously described in [6 (link),35 (link),36 (link)]. Briefly, after 5 days of activation, for the last 24 h, we added in the wells a mix of radioactive and nonradioactive palmitate coupled to BSA (2:1 ratio; 30 μM Na-palmitate, 15 μM fatty-acid-free BSA and 10 μCi (0.83 μM 9,10-³H-palmitic acid (Perkin Elmer)). After a 24 h additional incubation, 100% trichloroacetic acid (10% final) was added to the cell suspensions, and proteins were precipitated. After centrifugation, NaOH (final concentration 0.75 M) was added to the supernatant to increase pH to 12. Subsequently, 400 μL of supernatant was applied to ion-exchange columns (Dowex 1 × 2–400 resin), and ³H₂O was recovered by eluting with 4 mL of H₂O. A 0.75 mL aliquot was then used for scintillation counting. Results were expressed as CPM (counts per minute) per 10⁶ cells.