Supelco wax

To analyze the fatty composition of RAW 264.7 cells and algal biomass, transmethylation of fatty acids was performed by incubating polar lipids or freeze-dried biomass in dry methanol containing 2% (v/v) H2SO4 at 80 °C for 1.5 h under an argon atmosphere and continuous stirring. Pentanoic acid (C15:0; Sigma–Aldrich) was added as an internal standard. FAMEs were quantified on a Trace GC ultra (Thermo, Italy) equipped with a flame ionization detector and a programmed temperature vaporizing (PTV) injector. The detector temperature was fixed at 280 °C, and helium was used as a carrier gas. The PTV injector was programmed to increase the temperature from 40 °C at the time of injection to 300 °C at time of sample transfer. Separation was achieved on a fused silica capillary column (Supelco-WAX, 30 m × 0.32 mm). FAMEs were identified by co-chromatography with authentic standards (Sigma–Aldrich, Rehovot, Israel) [26 (link)].

IT-SPME was essentially performed as described in our previous works [31 (link),32 (link)]. A GC capillary column (60 cm × 0.32 mm i.d.) as an extraction device was connected between the injection needle and injection loop of the autosampler. The capillary column was threaded through a 1/16 inch polyetheretherketone (PEEK) tube with a length of 2.5 cm long and an inner diameter of 330 μm and connected using standard 1/16 inch stainless steel nuts, ferrules, and connectors. CP-Sil 5CB (100% polydimethylsiloxane, film thickness 5 μm), CP-Sil 19CB (14% cyanopropyl phenyl methylsilicone, film thickness 1.2 μm) (Varian Inc., Lake Forest, CA, USA), Supelco-Wax (polyethylene glycol, film thickness 1.0 μm), Supel-Q PLOT (divinylbenzene polymer, film thickness 17 μm), and Carboxen 1006 PLOT (carbon molecular sieve, film thickness 15 μm) (Supelco, Bellefonte, PA, USA) were used to compare extraction efficiencies. Extraction, desorption, and injection parameters were programmed by the autosampler software (Table S1) [31 (link),32 (link)].

Liver samples used for fatty acid analyses were kept frozen at −80°C and then lyophilized overnight. Total lipids were extracted from the powdered samples with the method of Bligh and Dyer (30 (link)) and evaporated to dryness under N₂ flow. Separation of total lipid extracts into neutral and polar lipid fractions was performed by sequential elution with chloroform and methanol on silica gel cartridges (Bond- Elut, USA). Polar and neutral fractions were then transmethylated, and fatty acid methyl esters (FAMEs) were extracted as described in Nayak et al. (31 (link)). A GC analysis of FAMEs was carried out on a Trace Ultra gas chromatograph (Thermo Fisher Scientific, USA) equipped with a flame ionization detector, a programmable temperature vaporization injector and a capillary column (SupelcoWAX, Sigma-Aldrich, USA) as described in Nath et al. (14 (link)).