Free fatty acid extraction and measurement
Cell culture samples (20 mL; three replicates for each sample) were centrifuged at 8000 rpm for 10 min to separate the cell-associated fatty acids from the extracellular fatty acids. Fatty acid extraction was performed as previously described [11]. The fatty acids extracted from the supernatant were analyzed through gas chromatography–mass spectrometry (GC-MS) by using a 5975 C Series MSD and Agilent 6850 equipped with an HP-5 MS column (30 m × 0.32 mm; film thickness of 0.25 mm). Helium was used as a carrier gas. The temperatures of the injector and the detector were 250 °C and 280 °C, respectively. The GC elution conditions were as follows: 100 °C as the starting temperature for 5 min, 15 min ramp to 250 °C, and 250 °C held constant for 5 min. All of the samples were spiked with pentadecanoic acid (C15) as an internal standard. The growth of the cells and the analysis of the fatty acid products were repeated thrice.

Results