PMC:3216505 / 16256-18157
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3216505","sourcedb":"PMC","sourceid":"3216505","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3216505","text":"Giant Unilamellar Vesicles\nGUVs were produced by a modification of the electroformation method24. The ‘Major-Minor mix' of lipids8 was prepared, consisting of the phospholipids DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, 51mol%), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 22 mol%), DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], 8 mol%), DOPA (1,2-dioleoyl-sn-glycero-3-phosphate, 5 mol%), soy PI (L-α-phosphatidylinositol, 8 mol%), porcine brain PI4P (L-α-phosphatidylinositol-4-phosphate, 2.2 mol%), porcine brain PI(4,5)P2 (L-α-phosphatidylinositol-4,5-bisphosphate, 0.8 mol%), CDP-DAG (1,2-dioleoyl-sn-glycero-3-(cytidine diphosphate), 2 mol%; all lipids from Avanti Polar Lipids, Alabaster, AL), fluorescent lipid analog (1 mol%, see below) and ergosterol (Sigma, St. Louis, MO; 20% ergosterol by weight, 80% phospholipids by weight). As fluorescent lipid analogs, DiIC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, Invitrogen, Carlsbad, CA; for single-color confocal microscopy), DiDC18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate, Invitrogen; for two-color confocal microscopy) and Texas-Red DHPE (Texas Red-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, Invitrogen) were used.\nLipids were combined in chloroform:methanol (2∶1, volume ratio) at 10 mg/ml total concentration and dried as a thin layer on indium-tin-oxide coated glass slides (Delta Technologies, Stillwater, MN). The slides were assembled to form a chamber by using 3 mm thick silicon spacers and holding the slides together with office clips. The chamber was filled with sucrose solution (540 mOsmol/kg) and electroformation was performed overnight using an approximately 3 Volt, 10 Hz sinusoidal voltage and copper conductive tape (3M, SPI supplies) for connections. The GUVs were harvested by sedimentation in an iso-osmolar glucose solution at 4°C.","divisions":[{"label":"title","span":{"begin":0,"end":26}},{"label":"p","span":{"begin":27,"end":1261}}],"tracks":[{"project":"2_test","denotations":[{"id":"22355536-9568718-137225160","span":{"begin":129,"end":130},"obj":"9568718"}],"attributes":[{"subj":"22355536-9568718-137225160","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93ecd7","default":true}]}]}}