D5 tag 48 0

Lipids from cell pellets were extracted using butanol:methanol (1:1) as extraction solvent. The extraction solvent included an internal standard mixture consisting of 17:0 LPC (lysophosphocholine), C14 LPE (lysophosphoethanolamine), C17 ceramide, C17 phosphocholine, C17 phosphoserine, C17:0 PG, C8-ceramide, DMPE (1,2-dimyristoyl-glycero-3-phosphoethanolamine), DMPG [1,2-dimyristoyl-glycero-3-phospho-(1′rac-glycerol)], DMPS (dimyristoyl-glycero-phosphoserine), 12:0 DG (1,2-dilauroyl-sn-glycerol), C8 GluCer (d-glucosyl-β1-1′-N-octanoyl-d-erythro-sphingosine), 24:0 SM (N-lignoceroyl-d-erythro-sphingosylphosphorylcholine), 24:1 SM (N-nervonoyl-d-erythro-sphingosylphosphorylcholine), and d5-TAG 48:0. All the standards were obtained from Avanti Polar Lipids (Alabaster, AL, USA) except for d5-TAG 48:0 that was obtained from CDN Isotopes. All standards were added to a final concentration of 100 ng/ml. One hundred microliters of the extraction solvent including the standards was added to 1 × 10⁷ cells in a vial containing the cell pellet. The mixture was vortexed for 15 s. The samples were then sonicated in a sonicator bath for 60 min at room temperature. The mixture was then centrifuged at 14,000g for 5 min. The supernatant was transferred to new tubes and kept at −80°C until the analysis.