Liposome preparation

To prepare liposomes, POPC:DOPE:NBD-PE were mixed at a volumetric ratio of 88:6:6 in glass tubes. Dried films were prepared by evaporating chloroform. Films were hydrated in buffer E to obtain 1 mM lipid solutions. The lipid suspension was incubated at room temperature for 1 h and mixed by vortex. Homogenously sized unilamellar vesicles were obtained using a mini-extruder set in combination with polycarbonate filters with a pore size of 400 and 100 nm (Avanti). To separate liposomes from NBD-PE, the liposome solution was centrifuged at 125,000 g for 1 h at 4°C and the supernatant was removed carefully. Then, buffer E was added and liposomes were analyzed using a Zetasizer Nano S (Malvern Instruments) as described previously (Kawamata et al., 2022).