Lipidomic analysis was performed at the University of California Los Angeles (UCLA) Lipidomic Core. A modified Bligh and Dyer extraction (93 (link)) was carried out on HAEC lysates or conditioned medium aliquots. Before biphasic extraction, a standard mixture of 75 lipid standards (Avanti, 330820, 861809, 330729, 330727, and 791642) was added to each sample. Following two successive extractions, pooled organic layers were dried down in a Thermo SpeedVac SPD300DDA using ramp setting 4 at 35°C for 45 min with a total run time of 90 min. Lipid samples were resuspended in 1:1 methanol:dichloromethane with 10 mM ammonium acetate and transferred to robovials (Thermo Fisher Scientific, 10800107) for analysis.
Samples were analyzed by direct infusion on a Sciex 5500 with Differential Mobility Device (DMS) (comparable to Sciex Lipidyzer platform) with a targeted acquisition list consisting of 1450 lipid species across 17 subclasses. The DMS was tuned with EquiSPLASH LIPIDOMIX (Avanti, 330731). Data analysis was performed with in-house data analysis workflow. Instrument settings, multiple reaction monitoring (MRM) lists, and analysis method are previously described (94 (link)). Quantitative values were normalized to cell counts or medium volume.
Samples were analyzed by direct infusion on a Sciex 5500 with Differential Mobility Device (DMS) (comparable to Sciex Lipidyzer platform) with a targeted acquisition list consisting of 1450 lipid species across 17 subclasses. The DMS was tuned with EquiSPLASH LIPIDOMIX (Avanti, 330731). Data analysis was performed with in-house data analysis workflow. Instrument settings, multiple reaction monitoring (MRM) lists, and analysis method are previously described (94 (link)). Quantitative values were normalized to cell counts or medium volume.
