Triolein

Methanol, chloroform, dichloromethane, and acetonitrile (Fisher) were all of mass spectrometry grade. Sodium formate and HCl were from Sigma, and TMS-diazomethane (TMS-DM, 2.0 M in hexanes) from Sigma-Aldrich and Acros. Lipid standards were ammonium salts of 1-heptadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate) [17:0-20:4 PI(4,5)P₂] Avanti Polar Lipids, LM1904; 1-heptadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1′-myo-inositol-4′-phosphate) [17:0-20:4 PI(4)P] Avanti Polar Lipids, LM1901; and 1-heptadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1′-myo-inositol-3′,4′,5′-trisphosphate) [17:0-20:4 PI(3,4,5)P₃] Avanti Polar Lipids, LM1906, Trimyristin (14:0, 14:0, 14:0), Tripalmitin (16:0, 16:0, 16:0), Triolein (18:1, 18:1, 18:1) Trilinolein (18:2,18:2,18:2), Tristearin (18:0,18:0,18:0), Triarachidin (20:0,20:0,20:0), Triarachidonin (20:4,20:4,20:4) (NuChek-Prep, Inc. Elysian, MN).

All phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Triolein was purchased from Nu-Chek-Prep (Elysian, MN, USA). Pure lipid was dissolved in 2:1 chloroform:methanol (>99% purity, Thermo Fisher Scientific, Waltham, MA) at a concentration of ~0.1 mM to prepare lipid stocks. Lipid films were made in a borosilicate glass tube by drying a specific volume of lipid stock solution(s) under a stream of nitrogen. The films were kept under vacuum overnight to remove residual traces of organic solvent and stored at −20 °C. Lipid films were rehydrated with 4 mL HPLC-grade water and vortexed for ~30 s. After vortexing, the mixture was put through five rounds of rapid freeze-thaws. This mixture was then extruded through a 200 nm and 100 nm filter following standard procedure (T&T Scientific, Knoxville, TN, USA). The size of the resulting vesicles was measured using DLS (differential scanning calorimetry, Horiba DLS 7100, SZ-100 series) to be between 50–250 nm. We found no significant difference between lipid adsorption to the oil surface with these size differences.