1 14c palmitic acid

[1-¹⁴C] palmitic acid (Perkin Elmer, Waltham, MA) or palmitic acid (Sigma) was resuspended in α-cyclodextrin (Sigma, 20 mg/ml in 10 mm Tris, pH 8) to obtain a 12 μCi/ml solution. HUVECs grown fully confluent in 35 mm dish were incubated with the indicated stimuli for the indicated time and then 1.2 μCi/ml [1-¹⁴C] palmitic acid was added for 4h in the presence or absence of stimuli. For the siRNA study, transfected HUVECs were seeded the day before the assay then incubated with [1-¹⁴C] palmitic acid for 4h. After 4h, the lids of the cell culture plates were covered inside with Whatman paper and saturated with 5 m NaOH. Addition of 0.5 N perchloric acid triggered the release of CO₂ that was captured in the Whatman paper and analyzed in a scintillation counter (MicroBeta TriLux, Perkin Elmer). For the acid soluble metabolites, 1 ml of medium was recovered, incubated with 200 μl 4N KOH, 30 min at 60 °C to hydrolyze the acyl-CoA, esters and acidified with 300 μl 1 m NaC₂H₃O₂ and 200 μl 3N H₂SO₄. After spinning, 300 μl of the supernatant were mixed with 5 ml of a 2:1 solution of chloroform/methanol to allow phase separation. The upper aqueous phase, where the acid soluble metabolites (ASM) coming from palmitic acid oxidation are dissolved, was incubated with scintillation fluid and analyzed in a scintillation counter. The values obtained were normalized by cell number.