1 2 dipalmitoyl sn glycero 3 phosphoglycerol

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Cholesterol was obtained from JT Baker (Avantor Performance Materials, Inc., Center Valley, PA, USA) and phosphate-buffered saline (PBS) at pH 7.2 was purchased from Invitrogen (Life Technologies, Carlsbad, CA, USA). GLA-LS was manufactured by combining 3.6 g DPPC, 1.0 g cholesterol, and 0.4 g DPPG in 20 mL of chloroform:methanol:water (65:35:8), which was then evaporated using a BUCHI Rotovapor R-114 (BUCHI, Flawil, Switzerland). The dried components were rehydrated in 100 mL PBS (pH 7.2), then sonicated in a VWR 75D (VWR International, Radnor, PA, USA) or Crest Powersonic CP230D (Crest Ultrasonics Corp, Trenton, NJ, USA) sonicating water bath at room temperature until all the solids were removed from the sides of the glass container. The mixture was then transferred to the Microfluidics M110P (Microfluidics, West-wood, MA, USA) for high-pressure homogenization at 10,000 psi for 12 passes. The stock liposome formulation contained 18 mg/mL DPPC, 2 mg/mL DPPG, and 5 mg/mL cholesterol, and was stored at 5°C. Liposomes were diluted 2.5-fold in PBS prior to cryo-TEM experiments.