J w cp sil 88

The total lipid content of the yeast cell was determined by an extraction using petroleum ether as the organic solvent. After filtration, the lipid residue was weighed after solvent evaporation. The fatty acid characterization was analyzed through fatty acid methyl ester (FAME) by transesterification with methanol. Briefly, 25 mg of the extracted yeast oil was placed in a test tube for mixing with 0.5 M methanolic NaOH 1.5 mL. Then, oxygen was removed by feeding of nitrogen gas and was heated for approximately 2 min at temperature 80–90 °C. Subsequently, it was left to cool, and 2 mL BF₃ solution was added. The mixture was further heated for 30 min and was shaken periodically. Then, it was mixed with 1 mL of iso-octane and 5 mL of 36% NaCl solution. After phase separation, the upper layer was collected, and the raffinate was extracted again with the same amount of iso-octane. The sample of 1 µL was injected to a GC (Agilent 7890A, USA) equipped with a FID detector. The GC column was Agilent’s J&W CP-Sil 88 (100 m × 0.25 mm × 0.2 µm) which provided a great efficiency and resolution for FAME compounds. The injector temperature was 240 °C, split 50:1, and the detector temperature was 250 °C, respectively.

The total lipids were extracted from BMECs in a 10 cm culture dish using 2 mL of 2.5% (vol/vol) sulfuric acid/methanol in a 15 mL glass tube with 200 μL of C11:0 fatty acid (Sigma, Shanghai, China) as an internal control. After ultrasonication for 10 min, the glass tubes were incubated at 80 °C for 60 min. After cooling the tubes to room temperature, 2 mL of 0.1 M HCl and 800 μL of hexyl hydride were added, and the glass tubes were vortexed for 30 s, followed by centrifugation for 5 min at 900× g and 4 °C. The supernatant was collected in a 2 mL silicified tube with 0.5 g of water-free sodium sulfate. After an acute shock, the tubes were centrifuged for 3 min at 13,800× g and 4 °C, and the liquid supernatant was collected and filtered with a 0.22 μm aqueous-phase membrane filter for fatty acid analysis through 7890A gas chromatography (Agilent Technologies, Shanghai, China) with an JW CP-Sil 88 (100 m × 0.25 mm × 0.20 μm, CP7489) column (Agilent Technologies, Shanghai, China). The amount of each fatty acid was determined. The results were normalized to the protein content.