Ovine cholesterol

The cloned gene of human serum amyloid A (SAA1, Cat# hMU009432) was obtained from the Human Gene Bank of Korea (Daejeon, Korea). The pET30a(+) expression vector and E. coli BL21 (DE3) were purchased from Novagen (Madison, WI, USA). The restriction enzymes were acquired from New England BioLabs (Beverly, MA, USA). Palmitoyloleoyl phosphatidylcholine (POPC, #850457P), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, #850345), and cholesterol (ovine, #700000P) were supplied by Avanti Polar Lipids (Alabaster, AL, USA). Sodium cholate (#C1254) was purchased from Sigma (St Louis, MO, USA).

Egg phosphatidylcholine (PC) and cholesterol (ovine) were purchased from Avanti Polar Lipids (Alabaster, AL). Liposomes were prepared using a lipid film hydration technique (42 (link)). Briefly, 13 mM of PC_100% or PC_70%Cholesterol_30% (molar ratios) were dissolved in a chloroform:methanol (5:1 ratio) solvent and dried under reduced pressure (50 mBar, 1 h at 45°C) to form a thin film. This film was rehydrated in sucrose-Tris buffer [280 mM sucrose, 10 mM Trizma-hydrochloride (pH 7.4), at 45°C for 1 h with agitation. The resulting liposome suspension was extruded sequentially through polycarbonate filters with pores of 400, 200, and 100 nm in diameter (Nucleopore Corp., Pleasanton, CA) using a handheld extruder (Mini-extruder; Avanti Polar Lipids) to produce a solution of unilamellar phospholipid vesicles of uniform size. Liposomes were labeled exclusively in the outer bilayer leaflet with di-8-ANEPPs, as described previously (27 (link)). Briefly, liposomes were incubated for 1.5 h at 37°C in the presence of di-8-ANEPPs (Thermo Fisher Scientific, Waltham, MA) dissolved in ethanol while protecting from light.

Lipids 1,2-diphytanoyl-sn-glycero-phosphocholine (DPhPC 4ME16:0PC) and cholesterol (ovine) were obtained from Avanti Polar Lipids (Alabaster, AL, USA) and mixed in chloroform to the desired mole percentage, 95% DPhPC and 5% cholesterol. SUVs were formed following the previously described [25]. Briefly, lipids were reconstituted in chloroform in a glass vial and the chloroform was evaporated until a thin lipid layer is deposited on the bottom of the glass vial. The lipid layer is then placed under vacuum, −23 inhg, for >4 hours. Lipids are then rehydrated in 1ml of diH2O overnight at 60°C. The following day, lipids are vortexed and then passed through a 100 nm polycarbonate filter with the Mini-Extruder (Avanti Polar Lipids, Alabaster, AL USA) 7 times and stored at 4°C for two weeks. SUVs with or without channel incorporated are dried onto indium tin oxide coated glass slides from Nanion Technologies (Munich, Germany). The dried slides are placed on the Vesicle Prep Pro (Nanion Technologies) with a rubber o-ring and 300 mM sucrose. GUVs are formed using the standard program. Upon GUV formation, vesicles are used same day.