Fatty Acid Extraction and Methylation

To 2.5-mL samples of cell culture (three replicates for each culture at each sampling time), 5 μL of 10 mg mL⁻¹ heptadecanoic acid (Fluka, Buchs, Switzerland) dissolved in ethanol and 50 μL of 10 mg mL⁻¹ pentadecanoic acid (Acros Organics, Geel, Belgium) dissolved in ethanol were added as internal standards. Next, 100 μL of glacial acetic acid and 5.0 mL of a 1:1 (v/v) chloroform/methanol mixture were added (Bligh and Dyer, 1959 (link)). The samples were inverted several times, vortexed vigorously, and centrifuged. The aqueous layer and cell debris were removed by aspiration and the chloroform layer was stored at −80°C until further processing. To methylate the fatty acids, the chloroform layer was thawed and evaporated under a nitrogen stream. Residual water was removed by lyophilization for approximately 1 h. To the dried residue, 0.5 mL of 1.25 M HCl in methanol (Fluka) was added, and the reaction was incubated overnight (14–16 h) at 50°C. The reaction mixtures were quenched by the addition of 5 mL of 100 mg mL⁻¹ aqueous NaHCO₃ (Sigma-Aldrich Corp., St. Louis, MO), and fatty acid methyl esters were extracted twice into 0.5 mL of hexane. The hexane layers were collected for analysis.