Preparation of plain SLB substrates for live-cell overlay experiments

Twenty-five-millimeter-diameter 1.5H cover glasses were cleaned in Piranha solution and stored in distilled water. Cover glasses were plasma-cleaned for 3 min just before incubating facedown on 30 μl of liposome suspension for 3 min. The cover glasses are assembled into Attofluor cell chambers while immersed in water and then exchanged into TBS. The surface was blocked with 0.05% BSA for 1 hour at 37°C. The composition of lipids used in plain lipid bilayers was 1% Ni-NTA-DOGS in DOPC for HRB binding experiments and 1% Ni-NTA-DOGS and 4% TR-DHPE in DOPC for hemifusion detection experiments.