Cholesterol ester

For T cell coculture assay, T cells were isolated by the EasySepTM Mouse T cell Isolation Kit (STEMCELL). Isolated T cells were cultured with conditional medium collecting from Hepa1-6 cells, supplied with mouse T-activator CD3/CD28 Dynabeads (Invitrogen) and mouse IL-2 (Biolegend, 10ng/ml) for 72 hours. Human peripheral blood CD8⁺ T cells were obtained from STEMCELL (#70027). Human CD8⁺ T cells were co-cultured with conditional medium collecting from different NASH-HCC cells, with human T-activation CD3/CD28 Dynabeads (Invitrogen) and human IL-2 (R&D Systems, 10ng/ml) for 72 hours, with or without cholesterol (Sigma), cholesterol ester (Sigma) or β- cyclodextrin (Sigma). Quantification of IFN-γ ⁺ and GZMB⁺ CD8⁺ T cells (among CD8⁺ T cells) from different groups was conducted. T cells were stimulated with PMA, monensin, and ionomycin for 4 hours. IFN-γ or GZMB-producing cells were determined by flow cytometry.

Commercial BM powder was kindly donated by Reny-Picot (Asturias, Spain). The sample was kept frozen at −35 °C until the analysis.
All solvents were at least HPLC grade and MS grade when available. Chloroform, hexane, methanol, isooctane, isopropanol, ammonium hydroxide, arsenic formate, and acetonitrile were purchased from LABSCAN (Dublin, Ireland). methanol (F.C.C.) ADITIO for industrial food use, sea sand, potassium hydroxide, and sodium carbonate were from PANREAC (Barcelona, Spain). Formic acid (98%) and triethylamine (99.5%), the TAG standards trinanoin and tridecanoin, the FFA standards pelargonic (C9), tridecanoic (C13), myristic (C14), palmitic (C16), estearic (C18), arachidonic (AA, 20:4), eicosapentaenoic (EPA, 20:5), and docosahexaenoic (DHA, 22:6) acids, the sterols 5α-cholestane, cholesterol (CHOL), cholesterol ester (CE), desmosterol, campesterol, β-sitosterol and lanosterol, monostearin, diolein and tripalmitin, as well as PI, PS, PE, SM, PC, and N-oleoylethanolamine standards were from Sigma-Aldrich (St. Louis, MO, USA). Reference samples with known composition as butter fat BCR-164 and BCR-519 (EU Com 121 missions; Brussels, Belgium) were from Fedelco Inc. (Madrid, Spain).