The inner aqueous (IA) solution of
GUVs consisted of a PBS buffer (10 mM phosphate buffer, 2.7 mM KCl,
and 137 mM NaCl) at pH 7.4, in addition to 200 mM sucrose and 15%
v/v glycerol. The outer aqueous solution additionally contained 50
mg mL–1 of Kolliphor P-188 (Sigma-Aldrich, U.K.)
as per standard OLA protocols.49 (link) Lipids
were purchased from Sigma-Aldrich in powder form and were dissolved
in 100% ethanol to a final concentration of 100 mg mL–1. The lipid/octanol phase (LO) was prepared by diluting the lipid
stock in 1-octanol to a 4–5 mg mL–1 concentration.
A lipid mixture of 3:1 ratio 1,2-dioleoyl-sn-glycero-3-phosphocholine
with 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)
sodium salt was used to mimic the anionic charge of typical bacterial
membranes.23 (link) 16:0 Liss Rhod PE (Avanti
Lipids, 0.5 mg mL–1 in ethanol) was doped to label
the LO phase. To monitor dye leakage from GUVs, HPTS (8-hydroxypyrene-1,3,6-trisulfonic
acid, Thermo Fisher) was diluted into the IA phase (50 μM).
GUVs consisted of a PBS buffer (10 mM phosphate buffer, 2.7 mM KCl,
and 137 mM NaCl) at pH 7.4, in addition to 200 mM sucrose and 15%
v/v glycerol. The outer aqueous solution additionally contained 50
mg mL–1 of Kolliphor P-188 (Sigma-Aldrich, U.K.)
as per standard OLA protocols.49 (link) Lipids
were purchased from Sigma-Aldrich in powder form and were dissolved
in 100% ethanol to a final concentration of 100 mg mL–1. The lipid/octanol phase (LO) was prepared by diluting the lipid
stock in 1-octanol to a 4–5 mg mL–1 concentration.
A lipid mixture of 3:1 ratio 1,2-dioleoyl-sn-glycero-3-phosphocholine
with 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)
sodium salt was used to mimic the anionic charge of typical bacterial
membranes.23 (link) 16:0 Liss Rhod PE (Avanti
Lipids, 0.5 mg mL–1 in ethanol) was doped to label
the LO phase. To monitor dye leakage from GUVs, HPTS (8-hydroxypyrene-1,3,6-trisulfonic
acid, Thermo Fisher) was diluted into the IA phase (50 μM).
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