The dehydration and rehydration
method79 (link),80 (link) was used to prepare the lipid suspensions.
Briefly, lipids (99 wt %) and lipid-conjugated fluorophores (1 wt
%) were dissolved in chloroform to a final concentration of 10 mg/mL
(cf.Table S1 for a detailed list of lipid
types and membrane compositions). 300 μL of this mixture was
then transferred to a 10 mL round-bottom flask, and the solvent was
removed in a rotary evaporator at reduced pressure (20 kPa) for 6
h to form a dry lipid film. The film was rehydrated with 3 mL of PBS
buffer (5 mM Trizma Base, 30 mM K3PO4, 3 mM
MgSO4·7H2O, 0.5 mM Na2EDTA,
pH 7.4 adjusted with 1 M H3PO4) and stored at
+4 °C overnight to allow the lipid cake to swell. The sample
was then sonicated for 25 s at room temperature, leading to the formation
of multi- and unilamellar giant vesicular compartments. For sample
preparation, 4 μL of the lipid suspension was desiccated for
20 min, and the dry residue was subsequently rehydrated with 0.5 mL
of HEPES-Na buffer containing 10 mM HEPES buffer and 100 mM NaCl,
adjusted to pH 7.8 with 5 M NaOH. The lipid suspension after rehydration
typically contains a few unilamellar vesicles in addition to MLVs
and the hybrid vesicles each comprising a unilamellar vesicle attached
to an MLV. The lipid suspension was thereafter transferred onto a
solid surface submerged in HEPES-Na buffer with addition of 4 mM CaCl2 (pH 7.8 adjusted with 5 M NaOH). The lipid vesicles in the
suspension spontaneously attach to the solid surface.
method79 (link),80 (link) was used to prepare the lipid suspensions.
Briefly, lipids (99 wt %) and lipid-conjugated fluorophores (1 wt
%) were dissolved in chloroform to a final concentration of 10 mg/mL
(cf.
types and membrane compositions). 300 μL of this mixture was
then transferred to a 10 mL round-bottom flask, and the solvent was
removed in a rotary evaporator at reduced pressure (20 kPa) for 6
h to form a dry lipid film. The film was rehydrated with 3 mL of PBS
buffer (5 mM Trizma Base, 30 mM K3PO4, 3 mM
MgSO4·7H2O, 0.5 mM Na2EDTA,
pH 7.4 adjusted with 1 M H3PO4) and stored at
+4 °C overnight to allow the lipid cake to swell. The sample
was then sonicated for 25 s at room temperature, leading to the formation
of multi- and unilamellar giant vesicular compartments. For sample
preparation, 4 μL of the lipid suspension was desiccated for
20 min, and the dry residue was subsequently rehydrated with 0.5 mL
of HEPES-Na buffer containing 10 mM HEPES buffer and 100 mM NaCl,
adjusted to pH 7.8 with 5 M NaOH. The lipid suspension after rehydration
typically contains a few unilamellar vesicles in addition to MLVs
and the hybrid vesicles each comprising a unilamellar vesicle attached
to an MLV. The lipid suspension was thereafter transferred onto a
solid surface submerged in HEPES-Na buffer with addition of 4 mM CaCl2 (pH 7.8 adjusted with 5 M NaOH). The lipid vesicles in the
suspension spontaneously attach to the solid surface.
