Preparation of Fluorescent Lipid Bilayers

Stock solutions (1–10 mg/mL)
of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS, Avanti Polar Lipids, Alabaster, AL, USA), and
ATTO 390-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
(Atto 390-DPPE, ATTO-TEC, Siegen, Germany) were prepared in chloroform. l-α-Phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P₂, brain porcine, Avanti Polar Lipids, Alabaster, AL, USA)
was freshly dissolved in chloroform/methanol/H₂O (10:20:8)
to a final concentration of 1 mg/mL. Lipid mixtures (0.4 mg) were
prepared in chloroform/methanol (10:1), and organic solvents were
evaporated with a nitrogen stream followed by 3 h in vacuum. The dried
lipid films were stored at 4 °C until needed.
Small unilamellar
vesicles (SUVs) were prepared by rehydrating a lipid film in spreading
buffer (50 mM KCl, 20 mM Na-citrate, 0.1 mM NaN₃, 0.1 mM
ethylenediaminetetraacetic acid (EDTA), pH 4.8),^{38 (link)} incubating for 30 min, subsequent vortexing (3 × 30
s at 5 min intervals), and a final sonification step for 30 min at
room temperature (cycle 4, 60%, Sonopuls HD2070, resonator cup; Bandelin,
Berlin, Germany). PtdIns[4,5]P₂ containing SUVs were used
immediately for the preparation of SLBs to avoid PtdIns[4,5]P₂ degradation.^{65 (link)}