Free cholesterol e and phospholipids c kits

Multilamellar vesicles were prepared with different lipid molar ratios (final concentration, 5 mg/mL) in 140 mM NaCl, 20 mM Tris, 5 μM EDTA, pH 8.0, as described previously^{60 (link)}. The aegerolysins were added to these vesicles at a 1/10 (w/w) ratio, and incubated for 30 min on a rotary shaker (600 rpm/min) at 25 °C, followed by centrifugation for 60 min at 60 000 × g and 4 °C. The supernatants contained the unbound aegerolysins, and they were transferred to vials, and precipitated with 20% trichloroacetic acid. After a 10-min incubation on ice, the precipitated proteins were sedimented by centrifugation at 14 300 × g for 12 min at 4 °C, and the pellets were washed twice with 300 μL ice-cold acetone. The fractions containing the free and bound aegerolysin proteins were diluted with an equal volume of nonreducing SDS sample buffer (20 mM Tris-HCl, pH 8.0, 5% [w/v] SDS, 2 mM EDTA, 0.1% [w/v] bromophenol blue), heated to 100 °C for 5 min, and applied to homogeneous 12% acrylamide gels. Proteins were stained with SimplyBlue SafeStain (Thermo Fisher Scientific, USA).
The lipid molar ratios and total lipid concentrations in lipid vesicle suspensions were determined colorimetrically using free cholesterol E and phospholipids C kits (Wako Pure Chemical Industries, Japan), and CPE concentrations were determined by quantification of the amine group^{65 (link)}.

Multilamellar vesicles were prepared with different molar ratios of lipids (final concentration, 5 mg/mL) in 140 mM NaCl, 20 mM Tris, 1 mM EDTA, pH 7.4, as described in^{33 (link)}. The lipid molar ratios and total lipid concentrations in lipid vesicle suspensions were determined colorimetrically using free cholesterol E and phospholipids C kits (Wako Pure Chemicals, Richmond, VA, USA). These suspensions of multilamellar vesicles were subjected to five freeze–thaw cycles and then extruded through 0.1 µm polycarbonate filters (Millipore, Burlington, MA, USA) at ~ 40 to 60 °C in order to prepare large unilamellar vesicles (LUVs). For the preparation of LUVs obtained from the lipids of the Sf9 cell line, the extraction of total lipids from this cell line was performed according to the protocol of Blight and Dyer^{34 (link)}. Small unilamellar vesicles loaded with calcein at the self-quenching concentration (80 mM) were prepared by sonication of multilamellar vesicles as described previously^{33 (link)}.