Preparation of Giant Vesicles
- Lipid solution (in chloroform) at ~25 mg/ml.
- Glucose and sucrose solutions.
- Teflon disk (~2 cm in diameter).
- Beaker (50 ml).
- Precision glass syringes.
- Vacuum pump.
- Hot plate.
- Wash teflon disk and beaker with detergent then water.
- Dry the teflon disk and polish it with sand paper.
- Wash the disk again with detergent, hot tap water, distilled water, and then a chloroform-methanol (2:1) solution.
- Dry the disk and the beaker thoroughly.
- Using a glass syringe, put about 30 ul of your lipid solution onto the teflon disk and spread it out. Place the disk into the beaker and cover the beaker with aluminum foil.
- Put the beaker under vacuum for several hours (~6 hours) to remove all the solvent.
- Lipid prehydration. Flow argon gas saturated in water vapor at a temperature above the transition temperature of the lipid over the disk for about 10 minutes.
- Final hydration. Add about 10 ml of distilled water or desired distilled sugar solution heated to 37C. We usually use a sucrose solution at 200 mM for hydration buffer.
- Place the beaker in a 37C incubator and incubate overnight.
On the following day, you will see "clouds" of vesicles. You can aspirate some with a Pasteur pipette and look at them directly under microscope. We usually dilute them in glucose with the same osmolarity as the sucrose solution we used for hydration. Since sucrose is heavier than glucose, the vesicles will sink to the bottom of the slide, facilitating observation under the microscope.
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