Biomod/2015/UTokyo-Komaba:EXPERIMENTS & RESULTS

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Materials


Hexane, cholesterol(Chol), stearylamine(SA), octadecyltriethoxysilane, Trion X-100, POPC,
Span 80(called sorbitan monooleate), Ca2+, fluorescence beads with negative charge, and Ca2+ indicator will be prepared for our experiment.

Methods

1.Preparation of solutions
As continuous oil phase, a hexane solution containing 3 wt% Span 80 and 0.1 wt% SA will be used.
The presence of SA enhanced the dispersability of the water droplets formed.
The hexane solution will be saturated with water by letting solution to be in contact with water at a volume ratio of 9:1(hexane solution: water), 4.5ml:0.5ml, for 30 min.
Then, the supernatant hexane solution will be removed and used, followed by centrifugation (5000 rpm for 8 min with a table centrifuge).
The supernatant hexane solution will be removed and used as continuous phase (named solution A).


As water phase, two types of solutions in different conditions will be used, Tris-HCl buffer (50mM, pH8) containing 1M Ca2+ and fluorescence beads with negative charge and Tris-HCl buffer (50mM, pH8) containing 0.4mM Ca2+ indicator.(named solution B1 and B2)


As hexane phase (named solution C), hexane solution containing 0.16 mg of lipid/ml at a POPC:Chol:SA molar ratio of 5:5:1 will be used.


2.Formation of W/O Emulsions by porous medium
Two types of water phases will be forced through porous medium into a continuous oil phase and water phases will be dispersed. Through the process, the size of water droplet will become the almost same as the size of hole in porous medium, about some μm. It is W/O emulsions.

3.Formation of Giant Vesicles(GVs)
In a first step, a monodisperse W/O emulsion containing solution B1 and B2 will be prepared by porous medium using Span 80 and SA as surfactant (see method section 1).
Subsequently, Span 80 and SA molecules that will cover the dispersed water droplets in the emulsion will be replaced by a bilayer-forming lipid mixture (solution C) at about -20 ℃ under conditions that keep the water droplets in a frozen state to avoid their coalescence.
The water droplets in the W/O emulsion will be frozen by cooling with liquid nitrogen; the surfactant replacement process will be performed at about -20 ℃, which will realize by cooling with ice mixed with salt.
The frozen water droplets (2.5µl of solution B1 and B2 (the sum amount of these solution is 5µl) in 500µl of W/O emulsion kept in micro tubes) will be precipitated for 30 min at -20℃, and 450µl of the supernatant will be removed by suction.
Then, 450µl of solution C will be added. The system will be mixed by micro pipette, followed by a sedimentation of the frozen water droplets for 30 min at -20℃.
During all this procedure, the water droplets will remain frozen. By repeating this replacement process another 3 times, the Span 80 concentration will be reduced to 0.01% of initial concentration, corresponding to 3 × 10-4 wt %.
After this surfactant replacement step, hexane will be evaporated by evaporator (from 500µl of W/O emulsion) at a temperature of -20℃ to keep water droplets frozen. After solvent evaporation, 200µl of 50mM Tris-HCl buffer (pH8), which will be kept at about -20℃, was added for lipid hydration and for the formation of GVs.
Finally, the sample will be warm up to room temperature.



Reference
S, Sugiura. T, Kuroiwa . T, Kagota. M,Nakajima. S, Sato. S, Mukataka. P, Walde. S, Ichikawa (2007) Novel Method for Obtaining Homogeneous Giant Vesicles from a Monodisperse Water-in-Oil Emulsion Prepared with a Microfluidic Device.Langmuir,24,4581-4588

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