Nfa galactocerebrosides

Large unilamellar vesicles (LUVs) and lipid nanotubes were prepared as previously described (Takei et al., 2001 (link)). For LUVs, 70% PS (Cat. No 840032C, Avanti), 10% biotinPE (Avanti) and 20% cholesterol (Avanti) were mixed and, for lipid nanotubes 40% NFA Galactocerebrosides (Sigma C1516), 40% PC (Avanti), 10% PI(4,5)P₂ (Calbiochem) and 10% cholesterol (Avanti) were mixed in 250 μl of chloroform in a glass vial (Mighty Vial No.01 4 ml, Maruemu Cat. No 5-115-03). Then chloroform was evaporated using slow-flow nitrogen gas to produce lipid a lipid film on the glass and then completely dried in a vacuum desiccator for 30 min. The dried lipid was rehydrated by water-saturated nitrogen gas followed by addition of 250 μl of filtered 0.3M sucrose for 2 hr at 37°C. The resultant LUVs and lipid nanotubes were passed through 0.4 μm- and 0.2μm- polycarbonate filters respectively 11 times using Avanti Mini extruder. The LUVs and lipid nanotubes (1 mg/ml of final concentration) were stored in dark at 4°C avoiding photooxidation.

Large unilamellar vesicles (LUVs) were prepared as previously described (46 (link)). For LUVs, 70% PS (840032C, Avanti), 10% biotinPE (870285X, Avanti), and 20% cholesterol (700000, Avanti) (w/v), and for lipid nanotubes, 40% NFA Galactocerebrosides (Sigma C1516), 40% PC (840051C, Avanti), 10% PI(4,5)P₂ (524644, Calbiochem), and 10% cholesterol (700000, Avanti) (w/v) were mixed and dissolved in 250 μl of chloroform and 75 μl methanol in Mighty Vial No.01 4 ml (5-115-03, Maruemu). Then the solvent was evaporated using slow-flow nitrogen gas to produce a lipid film on the glass and then completely dried in a vacuum desiccator for 1 h. The dried lipid was rehydrated by water-saturated nitrogen gas followed by addition of 250 μl of filtered 0.3 M sucrose for 2 h at 37 °C. The resultant LUVs and lipid nanotubes were passed through 0.4 μm- and 0.2 μm-polycarbonate filters, respectively, 11 times using Avanti Mini extruder (Merck). The LUVs and lipid nanotubes (1 mg/ml of final concentration) were stored at 4 °C in dark to avoid photooxidation.