Polycarbonate nucleopore membrane filter

Lipid stocks, DMPC, POPE/POPC/sphingomyelin/cholesterol (in proportions described above for LR), and TBE stored in 1:1 chloroform/methanol were dried under liquid nitrogen and vacuufuged overnight as described previously^{13 (link),43 (link),44 (link),46} and rehydrated with 15 mM 6-carboxy-fluorscein (6 -FITC) in 10 mM sodium phosphate buffer pH 8.00. The rehydrated lipid–dye mixture was subjected to 15 freeze–thaw cycles and subsequent extrusion with 200 nm polycarbonate nucleopore membrane filter (Whatman) with on a mini extruder to obtain dye-filled LUVs. The excess dye in the solution was separated from dye-filled LUVs using 7 kDa desalting columns preequilibrated with 10 mM sodium phosphate buffer pH 8.00 centrifuged at 500g for 30 s. The size of the LUVs was confirmed using DLS as mentioned below. The leakage of dye was confirmed by comparing the fluorescence intensity (λ_Ex: 490nm; λ_Em: 595nm) of intact dye-encapsulated liposomes and 2- to 3-fold increased intensity upon complete rupture of liposome upon addition of 0.2% Triton X-100.^{44 (link)} The percent dye leak is calculated by the difference between the dye leak intensity of LUVs with the protein and blank divided by the difference between the dye leak intensity of LUVs with Triton X-100 and blank LUVs as done previously.^{13 (link)}

The large unilamellar vesicles (LUVs) of ganglioside GM1 or POPC:POPG:Cholesterol:GM1 with a molar ratio of 7.65: 0.85: 1: 0.5 or total brain lipid extract (TLBE) (TLBE is mainly composed of PC/PE/PI/PS/PA with more than 55% of lipid constitute is unknown) were prepared by forming a nitrogen-dried lipid film from their chloroform stock solution followed by lyophilization overnight under high vacuum to get rid of the residual solvent. The lipid film was rehydrated in 10mM phosphate buffer, pH 6.0 and then vortexed well until it dissolved in the buffer. Finally the hydrated lipid solutions were extruded at least 23 times through a 100 nm polycarbonate nucleopore membrane filter (Whatman) mounted on a mini-extruder (Avanti Polar Lipids Inc. Alabaster, AL, USA) to obtain homogenous solutions of 100 nm sized LUVs^{39 (link), 40 (link)}. The homogeneity of the LUVs in solution was further confirmed through dynamic light scattering experiments.

LUVs were prepared as done previously.^{13 (link),41 (link)-44 (link)} DMPC, POPC/POPE/ sphingomyelin/cholesterol in 33/33/10/20% by weight (for LR), and TBE liposomes were constructed from a 1:1 chloroform/methanol solution of lipids stocks. The solution was gently dried under nitrogen flow and then placed in a vacufuge with desiccant overnight to further evaporate any residual solvent. The dried lipid film was then rehydrated with either a buffer solution (10 mM phosphate or 20 mM tris buffer, pH 8.0) or a buffer solution containing doping agent 10–50% (by weight) of GM1 or GM3 for DMPC or LR and TBE, respectively, to yield a final concentration of 1 mg/mL. The hydrated lipids were vortexed for 1–1.5 h at 37 °C and subjected to 15 freeze–thaw cycles with liquid nitrogen and water bath at ~50 °C. The resulting solution was extruded 25 times through a 200 nm (for LUVs) polycarbonate nucleopore membrane filter (Whatman) with a mini extruder to obtain unilamellar vesicles. The size of the vesicles was confirmed with DLS collected using a Zetasizer Nano S instrument (Malvern, Inc., Worcestershire, U.K.) as described below.