Preparation of giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) were produced by spontaneous swelling, following the procedures described in (17,19). For preparation of MPER-containing GUVs, vesicles and peptides were mixed at a 1:1000 peptide:lipid molar ratio for 15 min (25°C) before incubation with silica beads. Dried silica beads covered with lipid-peptide mixtures were collected and transferred to an 85 g/L sucrose buffer to induce spontaneous swelling of GUVs. Formed vesicles were transferred to a bovine-serum-albumin-blocked observation dish in an isosmotic 10 mM HEPES, 150 mM KCl (pH 7.4) buffer that already included 4E10 Fab to a final concentration of 3–20 nM for sFCS experiments and 200 nM for imaging experiments. GUVs and Abs were incubated for 15 min and subsequently imaged.

Atto 647-labeled Annexin A5 was purchased from Adipogen (Liestal, Switzerland). For the Annexin A5 experiments, GUVs were incubated with this protein in a 2 mM CaCl₂-HBS (HEPES-buffered saline) buffer.