9 10 3h palmitic acid

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.

CD4⁺ T cells from wild-type mice were isolated, activated and cultured in the absence or presence of GW0742 as described in the previous paragraph. For certain conditions, 50 μM etomoxir (Sigma) was added as well. After 48 hrs, media (including GW0742, etomoxir, or vehicle) was refreshed and 10 μl/well of a mix of radioactive and non-radioactive palmitate coupled to BSA (2:1 ratio; 15 μM fatty acid-free BSA (Sigma), 30 μM Na-palmitate (Sigma), and 10 μCi (0.83 μM [9,10-³H-palmitic acid (Perkin Elmer)) was added to each well. The radioactive and non-radioactive palmitate was coupled to BSA by first quickly adding the non-radioactive palmitate pre-heated at 70 °C to BSA pre-heated at 50 °C, followed by addition of the radioactive palmitate at this mix at 50 °C. After an additional 24-hr incubation, 100 % trichloroacetic acid (10% final) was added to the cell suspensions and protein was allowed to precipitate. 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 × 8–200, Sigma), and ³H₂O was recovered by eluting with 2.5 ml of H₂O. A 0.75-ml aliquot was then used for scintillation counting. Cell number was counted and the results were expressed as CPM × 10⁵/1 × 10⁶ cells.

After 48 h of transfection, LEC were supplemented with 100 µM unlabeled palmitate, 50 µM carnitine (Sigma-Aldrich), and 2 mCi/mL [9,10-³ H]-palmitic acid (Perkin Elmer) for 18 h in complete endothelial growth medium. Then, supernatants were collected into glass vials sealed with rubber stoppers. As a readout for the assay, ³H₂O was captured in Whatman paper soaked with H₂O over a period of 48 h at 37 C^{57 (link)}. Radioactivity was determined by liquid scintillation counting.