Atto488 dppe

The bilayers were formed by spin-coating a mixture of 1.27 mM DOPC dissolved in 1:1
chloroform/methanol solution with the addition of either 0.01 mol% KK114-DPPE, or 0.5
mol% of either DOPE-cap-biotin or DSPE-PEG-biotin. 25 µl of each
lipid solution were dropped on acid-cleaned round microscope cover glass
(Menzel-Glaser, 25 mm diameter, 1.5 mm thickness) and positioned on a spin-coater
(Chemat Technology). The cover glass was immediately spun at 3000–4000 rpm for 30 s.
This procedure induced evaporation of the solvent and formation of the SLB on the
cover glass, which remained stable for hours. The SLB-coated glass was then placed in
a microscopy liquid-tight chamber. Bilayers featuring biotinylated lipids were tagged
with the fluorescent gold nanoparticles in this phase, adding a solution of 3
µM tagged gold beads and incubating at room temperature for
30 min, which was followed by another washing step to remove unbound particles. In a
control experiment, we added 10 mM of free biotin (Life Technologies, ref. B20656)
seconds after the addition of the gold nanoparticles. Samples with KK114-tagged DPPE
did not undergo this passage. Atto488-DPPE (Atto-Tec) was used to independently
assess the presence of a bilayer region in the sample. The bilayers were kept
hydrated during FCS and iSCAT experiments with a 18 mM HEPES, 150 mM NaCl, pH 7.35
buffer.

Lissamine-rhodamin B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (RhB-PE) was purchased from Invitrogen (Copenhagen, Denmark). ATTO-647N-PE and ATTO-488-DPPE (ATTO-488 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) was purchased from ATTO-TEC GmbH (Siegen, Germany). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and Soy PC (catalog #840054) were from Avanti Polar Lipids (Alabaster, AL). Sodium cholate and all other chemicals used were obtained from Sigma-Aldrich (Copenhagen, Denmark). The buffer used was 10 mM phosphate buffered saline (PBS), 137 mM NaCl, and 2.7 mM KCl, pH 7.4.

To prepare the lipid stock solution, E. coli polar lipid extract (Avanti Polar Lipids, Inc.) was dissolved in water and sonicated for 30 min to make a 30-mg/mL aqueous suspension stock. The lipid stock solution was flash frozen in liquid nitrogen and stored at −80°C. To prepare a 2 mg/mL aqueous suspension stock of lipopolysaccharide, LPS from E. coli EH100 (Ra mutant; Sigma) was dissolved in water and sonicated for 30 min. The lipopolysaccharide stock solution was flash frozen in liquid nitrogen and stored in aliquots at −80°C.
To prepare the fluorescent lipid stock solutions for flow cytometry, E. coli polar lipid extract (Avanti Polar Lipids, Inc.) was dissolved in chloroform with 1% (molar ratio) ATTO-488 DPPE, ATTO-565 DPPE, or ATTO-647N DPPE (ATTO-TEC GmbH). Lipid mixtures were then dried into a film, hydrated, and lyophilized overnight. The lipid mixtures were then dissolved in water and sonicated for 30 min to make a 30-mg/mL aqueous suspension stock. The lipid stock solution was flash frozen in liquid nitrogen, and stored at −80°C.