Lipid were extracted as described earlier (Bartosova et al., 2021a (link)) with modifications. The cell pellets were snap-freezed by submerging them in liquid nitrogen for about 10 s and lyophilized overnight. Five milligram(s) of dried cell pellets were mixed with zirconium oxide beads (0.5 ± 0.01 g, Ø 1.4 mm) in 2 ml vials with 1 ml of a cold mixture of chloroform:methanol (1:2, v/v). The mixtures were homogenized with three bead-beating cycles at 6,500 rpm for 30 s with 15 s intermediate pause by a Precellys® 24 bead homogenizer with a Cryolys temperature controller (all Bertin Technologies SAS, Montigny-le-Bretonneux, France). Cold chloroform 333 μl were then added, followed by vortexing for 20 s. Phase separation was induced by adding 333 μl of water, followed by vortexing for 20 s. The phase separation was accelerated by centrifuging at 14,000 rpm for 5 min at 15°C. A lower chloroform layer containing lipids was collected and cleared of cell debris with a syringe filter with PTFE membrane, 0.2 um, Ø 13 mm (VWR, United States). For determination of total lipids, 300 μl of the lipid extracts were transferred to a pre-weighed glass vial, left in fume hood for evaporation and weighed after 2 days.
The remaining extracts were flushed with a stream of nitrogen and stored at −80°C in dark glass vials for analyses of lipid classes and species.
The remaining extracts were flushed with a stream of nitrogen and stored at −80°C in dark glass vials for analyses of lipid classes and species.
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