Lipids used were PC 16:0/18:0 and PG 16:0/18:1 PG at 10 mg/mL in chloroform (Avanti Polar Lipids). All experiments were performed protected from light. On day one, PC was either used alone or mixed with PG in a 90/10 ratio for a final volume of 50 μL. The resulting lipid mixes were vortexed for 10 s and centrifuged for 1 min at 16,100 × g. Lipids were then dried using a SpeedVac vacuum concentrator. The resulting dried lipids were resuspended in 100 μL of buffer A (HEPES 30 mM, KCl 200 mM, pH 7.4) or buffer B (MES 30 mM, KCl 200 mM, pH 5.5) and sonicated in a water bath sonicator for 10 min to help with the formation of multilamellar vesicles/liposomes (crude liposomes). The resulting solutions were stored overnight at 4 °C. The next day, liposomes were sonicated for 10 min and placed on ice. They were then diluted 1/100 in buffer A or B to prepare the working solution. For t0 conditions, lipids were extracted right away. For t60 conditions, lipids were extracted after incubation for 1 h at 37 °C with shaking. For t60 + LPLA2 conditions, lipids were extracted after incubation for 1 h at 37 °C with shaking in the presence of recombinant active human lysosomal phospholipase A2 (Echelon Biosciences, with a LPLA2: PG molar ratio of 1/10). All buffers and tubes used for lipid extraction were pre-chilled at 4 °C. Lipids were extracted from each initial 50 μL reaction volume using 900 μL chloroform:methanol (5:4). The resulting solution was vortexed for 10 sec and supplemented with 200 μL KCl 1 M. The resulting solution was vortexed for 30 s and incubated on ice for 1 min. This step was repeated twice. The solution was then centrifuged for 2 min at 16,100 × g. The organic layer was then transferred to a new tube, dried using a SpeedVac vacuum concentrator and stored at −80 °C.
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