Cf400 cu ul

Liposomes were prepared as previously described (Adachi et al., 2016 (link)) with some modifications. The lipids were mixed in a POPC:POPE:Liver PI:POPS:DPPA:rhodamine-DPPE ratio of 44:25:10:5:15:1 (% mol). In control liposomes without DPPA, the amount of POPC was increased to 59%. The lipids were dried under a flow of nitrogen gas for 5 min and further dried in a SpeedVac overnight. The lipid film was resuspended at a concentration of 10 mM in 150 mM KCl and 20 mM MES (pH 7.0), voltexed for 1 hour, and subjected to freeze-thaw cycles 5 times using dry ice and a 42°C heat block. Unilamellar liposomes were generated via extrusion through a nanopore membrane with a pore size of 400 nm; this process was repeated 21 times. The liposomes (50 μM lipids) were incubated with peptides (0, 0.1, 0.3 and 1 μM) in 150 mM KCl and 20 mM MES (pH 7.0) at room temperature overnight. For negative-stain EM, the samples (20 μl) were placed on EM grids (CF400-CU-UL, Electron Microscopy Sciences, Hatfield, PA, USA) that have been glow discharged by Quorum GloQube-D (Laughton, East Sussex, UK). After being incubated for 2 min, the grids were washed with 150 mM KCl and 20 mM MES (pH 7.0) three times, stained with 0.1% uranyl acetate and 0.04% Tylose for 30 sec twice and then dried. The samples were observed using a Hitachi 7600 transmission electron microscope.