Lipid components of DOPG, DOPC, DOPE, DOPC/DOPG (DOPG, 8–95%), DOPC/CL (3 : 1), DOPC/DOPE/CL (DOPC : DOPE = 2 : 1; CL (TOCL/TMCL), 5–50% of total lipids), or lipids separated from mitochondria were dissolved in chloroform in a flask. The chloroform solvent was removed with a rotary evaporator at 40 °C, forming a thin lipid film on the wall of the flask. Residual chloroform in the thin lipid film was completely removed by drying in vacuo for 3 h. The lipids were hydrated by addition of a buffer with desired pH (2.0–8.3) at 40 °C. The lipid solution was mixed with a vortex mixer for ∼2 min for complete dissolution of the lipids. Seven cycles of freeze-and-thaw were performed at –196 and 50 °C to obtain multilamellar vesicles (MLVs). MLVs were extruded 15 times through two stacked polycarbonate membrane filters (pore size, 100 nm) equipped in a Liposo Fast mini extruder (Avestin, USA) to adjust the diameter of LUVs to 100 nm.
Liposo fast mini extruder
Manufactured by Avestin
Sourced in United States
The Liposo Fast mini extruder is a compact laboratory device used for the extrusion of liposomes and other lipid-based materials. It features a small footprint and is designed for efficient vesicle formation.
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Lab products found in correlation
4 protocols using liposo fast mini extruder
1
Preparation of Large Unilamellar Lipid Vesicles
2
Preparation of Unilamellar Liposomes
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An appropriate amount of 1 or a mixture of 1 and 2 ([1] : [2] = 7 : 3 mol mol−1) was dissolved in chloroform. The solvent was evaporated under a gentle stream of nitrogen, followed by a period under vacuum to remove any traces of solvent. The resulting thin lipid films were hydrated on the wall of the vial above the phase transition temperature with an appropriate amount of phosphate buffer. The hydrated materials were subjected to eight freeze–thaw cycles (−195 and 50 °C) to give unilamellar vesicles, which were extruded 11 times through 0.05 μm pores using a LiposoFast miniextruder from Avestin (Ottawa, Canada) above the phase transition temperature. The resulting liposomes were uniform in size with a diameter of approximately 80 nm. The final lipid concentration was 3.0 mM.
3
Preparation of Unilamellar Lipid Vesicles
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An appropriate amount of a mixture of DMPC (6.00 μmol) and 1 or NNM-1 ([DMPC] : [1 or NNM-1] = 10 : 1 mol mol−1) was dissolved in chloroform. The solvent was evaporated under a gentle stream of nitrogen, followed by a period under vacuum to remove trace solvent. The resulting thin lipid films were hydrated on the wall of the vial above the phase transition temperature with an appropriate amount of water or D2O (1.5 mL). The hydrated materials were subjected to eight freeze–thaw cycles (−195 and +50 °C) to give unilamellar vesicles, which were extruded 11 times through 0.05 μm pores using a LiposoFast™ Mini-extruder (Avestin, Ottawa, ON, USA) above the phase transition temperature.
4
Liposome Formation and Extrusion Protocol
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Protein-free liposomes were prepared by extrusion using an Avestin mini-extruder [1] . The liposomes used to form bilayer membrane contained 63 mol% DOPC, 25 mol% DOPS, 2 mol% PI(4,5)P2, and 10 mol% DAG. The liposomes used for vesicle capture contained 68 mol% DOPC, 30 mol% DOPS, 2 mol% DOPE-Atto647N. Detailed description was included in the Supplementary Information.
Chloroform solutions of the lipid mixtures were dried with a nitrogen stream for 10 min, followed by vacuum drying for 1 hour. The thin lipid films were hydrated with 500 L buffer containing 50 mM HEPES (pH 7.4), 140 mM KCl, and 10% glycerol. The mixture was vortexed vigorously for 30 minutes at room temperature. The multilamellar liposomes were frozen in liquid nitrogen for 30 sec, and were then thawed in a water bath at 37 o C for 30 sec. This cycle was repeated eight times. Small unilamellar liposomes of ~100 nm were produced by extrusion through 100 nm polycarbonate filters using a Liposofast mini-extruder (Avestin) 21 times at room temperature.
Chloroform solutions of the lipid mixtures were dried with a nitrogen stream for 10 min, followed by vacuum drying for 1 hour. The thin lipid films were hydrated with 500 L buffer containing 50 mM HEPES (pH 7.4), 140 mM KCl, and 10% glycerol. The mixture was vortexed vigorously for 30 minutes at room temperature. The multilamellar liposomes were frozen in liquid nitrogen for 30 sec, and were then thawed in a water bath at 37 o C for 30 sec. This cycle was repeated eight times. Small unilamellar liposomes of ~100 nm were produced by extrusion through 100 nm polycarbonate filters using a Liposofast mini-extruder (Avestin) 21 times at room temperature.
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