Cellular fatty acid methyl ester analysis

Cells cultured in 100 mm plates were washed twice with PBS; total fatty acids from the cells were extracted using Folch’s solution (chloroform/methanol solution; 2:1, vol/vol) and converted to methyl esters using methyl esterification solution (BF3/methanol/hexane; 4.9:75.1:20, vol/vol). Chromatographic analysis was performed using gas chromatography system (GC-7980, Techcomp, Shanghai, China) with silica capillary column (SP^T-2560, 100 m×0.25 mm×0.20 μm film thickness; Supelco, Bellefonte, PA, USA). The oven was set at a temperature of 70°C for 1 min, increased to 100°C at a rate of 5°C/min, maintained at 100°C for 2 min, increased to 175°C at a rate of 10°C/min, maintained at 175°C for 40 min, further increased to 225°C at a rate of 5°C/min, and maintained at 225°C for 40 min. Sample or standard mixture of fatty acid methyl esters (1 μL; FAMEs; Supelco 37-component FAME Mix, Supelco, USA) was injected into the column, carried by nitrogen, and detected by hydrogen flame ionization detector. The FAME components of sample were determined by comparing retention time peak with the appropriate FAME standards. Individual fatty acids were expressed as a weight percentage of the total fatty acids analyzed.