1 2 dipalmitoyl sn glycero 3 phosphoethanolamine n lissamine rhodamine b sulfonyl rhod pe

Synthetic lipid components, including 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero3-(phospho-L-serine) (DOPS), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (PEG-PE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE), were purchased from Avanti Polar Lipids (USA). As indicators in the lipid mixing assay, the lipid analog fluorescent dyes DiI and DiD were used as FRET donor and acceptor dyes, respectively (Invitrogen, USA). Chloroform stock solutions were mixed, and the chloroform was removed under vacuum in a desiccator for 16 h. The molar ratio of POPC:DOPS:PEG-PE was 92:7:1, and 10^‐5 mol % Rhod-PE was added for the formation of the supported lipid bilayer (SLB). For an in vitro lipid mixing assay, the molar ratio of POPC:DOPS:Chol:DiI (or DiD) was 71:7:20:2. The lipid mixtures were then rehydrated in 25 mM HEPES buffer with 100 mM KCl (pH 7.4), and ten freeze-and-thaw cycles were performed using liquid nitrogen and a water bath at 37°C. The solution was passed through a mini extruder (Avanti Polar Lipids) ten times with a 100 nm polycarbonate filter (Whatman, UK). Next, extrusion was performed to obtain monodisperse unilamellar vesicles using a mini extruder (Avanti Polar Lipids) with a 100 nm polycarbonate filter (Whatman).