100 nm polycarbonate filter

Liposomes were prepared from POPC, DOPS, POPE, and brain PIP₂ (Avanti Polar Lipids) stored individually as chloroform stocks, except for brain PIP₂ (stored in 20:9:1 CHCl₃:MeOH:H₂O). The lipids were combined, the solvent was evaporated under a stream of nitrogen, and the films were dried under vacuum for at least 2 hrs. Films were rehydrated in reconstitution buffer (100 mM KCl, 25 mM HEPES pH 7.4) at a final concentration of 10 mM [lipid] and extruded at least 30 times through a single 100-nm polycarbonate filter (Whatman), yielding liposomes of ~100 nm in diameter. Lipid compositions included 25% DOPS, 30% POPE, and 45% POPC, with POPC replacing DOPS in the 0% DOPS formulations and PIP₂ replacing POPC in the 1–3% PIP₂ formulations.

All lipids and lipid extracts were purchased from Avanti Polar Lipids. To prepare unilamelar liposomes from E. coli and bovine liver lipids, polar extracts were hydrated at 5mg/ml with 20mM HEPES, 150mM NaCl (pH7.5) and then vortexed continuously for 5min at room temperature. To prepare unilamellar liposomes of defined lipid composition, Phosphatidylcholine (PC) was mixed with either phosphoinositide (PI), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), sulfatide, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), or Di[3-deoxy-D-manno-octulosonyl]-lipid A (Kdo2-Lipid A, KLA) at an 80% to 20% molar ratio. Lipids were hydrated at 5mM with 20mM HEPES, 150mM NaCl (pH7.5) and then vortexed continuously for 5min at room temperature. Liposomes were generated by extrusion of the hydrated lipids through a 100nm polycarbonate filter (Whatman) 31 times using a Mini-Extruder device from Avanti Polar Lipids Inc. The CL complemented bovine liver liposomes were made by dissolving 20mg of bovine polar liver extract in chloroform. Then 5mg of CL dissolved in chloroform was added, and vortexed continuously for 5 minutes. The lipids, dissolved in chloroform, were placed on a heat block set to 50°C and evaporated under a stream of nitrogen. The lipid film was then hydrated in HEPES buffer and extruded as before.

Phosphatidylcholine (PC) vesicles and PS-containing PC vesicles were prepared using previously described methods.^{23 (link)} Specific details are available in the on-line Supplementary Information. Briefly, large unilamellar vesicles (LUV, 100 nm) were made by passing a suspension of large multilamellar vesicles through a 100 nm polycarbonate filter (Whatman, GE Healthcare, Uppsala, Sweden) using a manual extruder (Avestin, Ottawa, Canada). Vesicles were stored at RT and used within 7 days of preparation.
The optimal concentration of LUV for blocking experiments was determined by the ability of the PS-containing vesicles to inhibit the binding of FITC-lactadherin to tBuOOH-treated RBC, compared to PC vesicles. Specific details are available in the on-line Supplementary Information. Based on these titrations, 1 mM concentration of LUV was selected as the optimal dose to distinguish specific inhibition by PS-PC vesicles from background inhibition by PC-only vesicles.
To test inhibition of RBC-EC adhesion by lipid vesicles, HUVECs were pre-incubated with a 1 mM suspension of LUV for 30 minutes at 37 °C. Lipid vesicles were added at 10 µM final concentration to the tBuOOH-treated RBCs immediately before flow perfusion commenced to ensure the presence of excess LUV during the perfusion experiment.