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