Lipid extraction and mass spectrometry quantification

Total lipids of T cells or eluted TCR-lipids complexes were extracted with the two-step Bligh and Dyer method. Briefly, mix and vortex T cells with chloroform/methanol (1/2) for 10 min. Then add chloroform with additional vortex, and water with final vortex. After centrifugation, transfer the lower phase to a new tube. Add chloroform to the upper phase to repeat the extraction procedure. Cholesterol-26,26,26,27,27,27-D6 and 5, 24-cholestadien-3β-ol sulfate sodium salt were added to the extraction as internal controls of cholesterol and cholesterol sulfate, respectively. Evaporate under nitrogen gas, the lipid extract was dissolved in 200 μl methanol. Cholesterol and cholesterol sulfate were separated by Accela 1250 HPLC system (Thermo Fisher Scientific) with BEH C18 column. The samples were quantified by TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific) at Stanford University mass spectrometry facility. The MS of cholesterol and cholesterol sulfate were operated in APCI and ESI mode, respectively.