Atto 647n 1 2 dioleoyl sn glycero 3 phosphoethanolamine dope

GUVs were composed of 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), cholesterol (both Avanti Polar Lipids, Alabaster, AL, USA), Atto 647N 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; Sigma-Aldrich Chemie GmbH, Darmstadt, Germany), and either FSL-A(tri) (function-spacer-lipid with blood group A trisaccharide; Sigma-Aldrich Chemie GmbH, Darmstadt, Germany) and Ganglioside GM3 from bovine milk (Avanti Polar Lipids, Alabaster, AL, USA) at a molar ratio of 64.7:30:0.3:5 mol%.
GUVs were prepared by the electroformation method as earlier described [58 (link)]. Briefly, lipids dissolved in chloroform with a total concentration of 0.5 mg/mL were spread on indium tin oxide (ITO-covered) glass slides and dried in a vacuum for at least one hour or overnight. Two ITO slides were assembled to a chamber filled with sucrose solution adapted to the osmolarity of the imaging buffer of choice, either HBSS (live-cell imaging) or PBS (GUVs only imaging). Then, an alternating electrical field with a field strength of 1 V/mm was implemented for 2.5 h at room temperature. Later, we observed the GUVs in chambers manually built as described [60 ].

GUVs were composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (both AvantiPolar Lipids, United States), Atto 647 N 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; Sigma-Aldrich), and one of the following glycolipids at a molar ratio of 64.7:30:0.3:5. The glycolipids are globotriaosylceramide (Gb3, Matreya), FSL-Gb3 (Function-Spacer-Lipid with globotriaosyl saccharide), and corresponding FSL-isoGb3 (Function-Spacer-Lipid with iso-globotriaosyl saccharide) synthetized as previously described^{84 (link),85 (link)}, FSL-A(tri) (Function-Spacer-Lipid with blood group A trisaccharide; Sigma-Aldrich), FSL-B(tri) (Function-Spacer-Lipid with blood group B trisaccharide; Sigma-Aldrich) or lactosylceramide (LC, Sigma-Aldrich).
GUVs were prepared by the electroformation method as earlier described⁸⁶ . Briefly, lipids dissolved in chloroform of a total concentration of 0.5 mg/mL were spread on indium tin oxid-covered (ITO) glass slides and dried in a vacuum for at least 1 h or overnight. Two ITO slides were assembled to create a chamber filled with sucrose solution adapted to the osmolarity of the imaging buffer of choice, either HBSS (live-cell imaging) or PBS (GUVs only imaging). Then, an alternating electrical field with a field strength of 1 V/mm was implemented for 2.5 h at RT. Later we observed the GUVs in chambers manually built as described⁸⁶ .

GUV were composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (both Avanti Polar Lipids, United States), Atto 647N 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; Sigma Aldrich), and purified porcine Gb₃ (Matreya, United States) at a molar ratio of 64.7: 30: 0.3: 5 mol%.
GUV were prepared using the electroformation technique as previously described (Madl et al., 2016 (link)). Briefly, lipids dissolved in chloroform were deposited on indium tin oxide (ITO)-coated glass slides (Präzisions Glas and Optik, Germany). After complete evaporation of chloroform solvent, lipids were hydrated with 300 mM sucrose in a pre-assembled chamber. GUV were formed by applying an alternating voltage (Vmax = 1.1 V) to the chamber for 2-3 h.
GUV were imaged on an inverted confocal microscope (Eclipse Ti-E, Nikon, Japan; with a Nikon A1R confocal laser scanning system, and 60× oil immersion objective, NA = 1.49). The software NIS-elements (Nikon) was used for image acquisition and ImageJ for analysis.