Protocol for Vesicle Formation
- Nitrogen tap + plastic tubing
- Desiccator connected to a vacuum
- 100ml glass bottle
- Sonicator with medium-sized probe
- Ice bath
- 25°C incubator
- Pipette + pipette tips (1000µl)
- 10ml dodecane
- 12.5µl 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
Preparing the lipid-oil suspension for the inner leaflet
- Place 125µl of the 20mg/ml DOPC solution in a 100ml glass bottle.
- With the plastic tubing and 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film.
- Put the bottle in a desiccator connected to a vacuum for 1h.
- Add 50ml of mineral oil to reach a final lipid concentration of 0.05mg/ml.
- Set the sonicator probe to pulse 1, timer at 30mins.
- Put the bottle containing the suspension in the ice bath.
- Secure the sonicator probe inside the bottle, and set the amplitude to a reading of 10 when it is sonicating.
- Sonicate the suspension for 30mins.
- Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil.
- Magnetic stirrer
- Centrifuge + 1-inch glass centrifuge tubes
- Pipette + pipette tips (200µl, 1000µl)
- 50ml glass tube
- 5ml syringe
- Long 16-gauge stainless steel needle
- 10ml ddH2O
- Tris buffer
Emulsifying the aqueous solution (while the interface settles)
- Separate about 5ml of the lipid-oil suspension into a glass container. This is for the interface preparation.
- Prepare a 10ml solution A with 100mM NaCl and 5 mM Tris buffer at pH 7.4.
- Prepare solution B by adding a suitable quantity of reporter to 1ml of solution A.
- Add 250µl of solution B to the 45ml lipid-oil suspension in mineral oil.
- Gently stir the mixture with a magnetic stir bar for 3h.
Preparing the interface (to be done while the emulsion is mixing)
- Place 2ml of lipid-oil suspension over 3ml of solution A in a 1-inch-diameter centrifuge tube.
- Leave for 2–3h for lipids to achieve the coverage of the interface surface.
Forming the vesicles
- Pour 100µl of the inverted emulsion over the interface.
- Centrifuge at 120g for 10min.
Collecting the vesicles
- Using a 5ml syringe with a long 16-gauge stainless steel needle, collect some of solution A.
- Expel some of the solution to remove all air from the syringe and needle.
- With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe.
- Gently recirculate the buffer several times.
- Aspirate most of the solution into the syringe, and remove the needle from the solution.
- Wipe the tip of the needle clean.
- Unload the vesicle suspension into its final container.
(Note: Use optical microscopy to check that the vesicles obtained arenot deformed or aggregated.)
- Time Required:
- The lipid-oil suspension preparation takes about 2h (with a 1h waiting period 15min into the procedure), before being left overnight.
- The remainder of the procedure takes another 4h, with one 2h waiting period after an initial 1h preparation.
- Total working time in the lab is around 3 hours.
- The original protocol uses anhydrous 99:1 dodecane:silicone oil solution instead of mineral oil.
- The original protocol uses POPC instead of DOPC phospholipids.
- The original protocol sonicates the suspension in a cleaning sonic bath for 30min.
- Do not use rubber tubing in the nitrogen evaporation. This emits debris into the lipids.
- This procedure should form around 10^9 vesicles with 1µm diameter.
- Use of salt in the solution A preparation may require osmolarity considerations.
- Use of GFP as a visual signal may require osmolarity considerations.
- The reporter in solution B is optional. The vesicles may be visible without it.
- The interface should settle for more than 2h, but less than 3h. More than 3h causes the lipids to clump.