• Lipid
• Chloroform
• Methanol
• 50-µl Glass Syringe
• 50-ml Beaker
• Parafilm
• Aluminum Foil
• Pasteur Pipette
• 0.2 or 0.45 µm Syringe Filter
• Disposable Syringe
• Nitrogen/Argon Gas
• Teflon Disk (~1/16" thick)-Cut a circular Teflon disk to fit in the bottom of a 50-ml beaker. Roughen each surface of the disk with medium grade sandpaper. Thoroughly clean each disk with detergent and rinse thoroughly with deionized water, methanol and then chloroform.

Method:

1. Prepare a lipid stock solution (1 mg/ml) in chloroform:methanol (2:1 v/v).
   
2. Place a 30 µl drop of stock lipid solution using a 50-µl glass syringe onto one side of a roughened Teflon disk. Quickly spread the lipid solution over the entire Teflon surface using the syringe needle. With the “lipid” side up, place the disk into a 50-ml beaker.
   
3. Remove the volatile organic solvents from the lipid by placing the beaker in vacuo for at least 2 hours. Light sensitive mixtures that contain polyunsaturated lipids should be protected from light using aluminum foil. Purge the vacuum with nitrogen or argon gas.
   
4. Cover the beaker loosely with Parafilm and prehydrate the lipid film with a warm (45-50 °C) water-saturated argon/nitrogen jet for 15 minutes.
   
5. Preheat the buffer to be encapsulated at a temperature greater than the main phase transition temperature of the highest melting lipid (> 10 °C). Using a disposable plastic syringe fitted with a 0.2 or 0.45 mm syringe filter, gently add the rehydration buffer onto the pre-hydrated lipid film. Add enough buffer to completely immerse the entire Teflon disk. Seal the beaker with Parafilm to prevent water evaporation and incubate for several hours above the main phase transition temperature of the lipid (> 10 °C). Typically, vesicles will form after the system is left to swell overnight.
   
6. During the incubation period, the lipid film will swell and strip away from the Teflon surface to form small “clouds” of vesicles. At this time, vesicles are ready for harvest and can be collected by gentle aspiration using a Pasteur pipette.

Reference:

1. Needham, D., T. J. McIntosh, and E. Evans. 1988. Thermomechanical and transition properties of dimyristoylphosphatidylcholine/cholesterol bilayers. Biochemistry 27:4668-4673.