Solution Compositions

OLA, as employed here, requires five different solutions to carry out an experiment: inner aqueous (IA), outer aqueous (OA), lipids in 1-octanol (LO), exit well solution (EX), and a feed solution (FE). IA, OA, and EX always contained 15% v/v glycerol, 150 mM KCl, 5 mM MgCl₂, and a pH-regulating buffer or a base, unless otherwise indicated. Additionally, 5 mM dextran and 5% w/v P188 surfactant were always present in IA and OA, respectively. A detailed list of solution compositions for various experiments can be found in Supplementary Table 1. For each experiment, the osmolarity of the aqueous solutions was calculated, and, if needed, the solutions were balanced by addition of glucose. LO was always prepared by mixing lipids (10% w/v in ethanol, 99.9% DOPC + 0.1% Rh-PE, molar ratio) with 1-octanol to a final concentration of 0.2% w/v. Exceptions were experiments that included charged lipids: for PIP₃, lipid composition was 99.5% DOPC, 0.4% PIP₃, 0.1% Rh-PE (molar ratio). To induce as little movement in the exit well as possible when adding the feed solution, we used a Hamilton syringe (7105, 5 μL volume capacity) for the stepwise addition of submicroliter volumes of feed by fusing a small droplet at the end of the needle with the surface of the exit well solution containing the liposomes (∼3 mm distance between surface and liposomes at the bottom of the well).