Total lipid was resuspended in butanol and separated by high performance thin-layer chromatography (HPTLC, Silica gel 60, Merck), as described previously [50 (link)]. HPTLC was migrated with the solvent system hexane/diethyl ether/formic acid (40:10:1, v/v/v, Sigma) and revealed under UV light after spraying with purimuline (1 × 10−3%, Sigma) solution in 80% acetone. Different lipids were identified by comparison with an authentic standard spotted on the same plate. The spots correlating to phospholipids, DAG, sterols, free fatty acids, and TAG were scraped off. An internal standard (C15:0 1 nmol) (Avanti Polar lipids) was added and the methanolysis was carried out by hydrogen chloride solution. All samples were incubated at 85 °C for 3 h (oven) [50 (link)].
A second extraction was realized by adding hexane (Sigma) and water. The supernatant was transferred. For sterols, the derivatization was made by adding BSTFA-TMCS (Sigma) and for the rest of lipids, hexane was added. All the samples were analyzed using GC-MS (Agilent). For lipidomic analysis, three independent experiments were performed in triplicate for each sample.
A second extraction was realized by adding hexane (Sigma) and water. The supernatant was transferred. For sterols, the derivatization was made by adding BSTFA-TMCS (Sigma) and for the rest of lipids, hexane was added. All the samples were analyzed using GC-MS (Agilent). For lipidomic analysis, three independent experiments were performed in triplicate for each sample.
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