Editing Liposome Protocol

Liposome Extrusion Protocol Day 1

    1. Prepare a starting concentration of 50 mM of lipids (e.g. 1:1 DOPC:DSPC + 30% cholesterol + 5% N-dod-DOPE).
   2. Use the rotary evaporator to evaporate the chloroform out; ~30 mins (90 RPM, use heat if necessary - see lipid with the highest Tm).  
   3. Leave in vacuum desiccator overnight OR in vacuum oven if it requires heat.

Day 2

    1. Dissolve lipid film by adding 1 ml of 7:3 vol/vol DMSO:EtOH to the flask.  Vortex until lipid film has visibly disappeared.
   2. Add rhodamine-dextran (in 4C fridge) at a concentration of 1 mg/ml to 3 ml of the buffer that will be used (e.g. PBS 1X).  Note: rhodamine is light-sensitive; all work must be done with the lights off.
   3. Add .3 ml of dissolved lipids to the buffer + rhodamine-dextran dropwise while stirring on a hot plate at 750 RPM.  (use heat again if it is required)  Note: use up .9 ml of dissolved lipids so prepare 3 batches of PBS + rhodamine-dextran; in the end you have 9 ml total to extrude.
   4. Wash the extruder and its parts with warm soapy water, rinse with DI water, and spray with 70% EtOH (except for O-rings and Tygon tubing).  Dry and make sure the mesh filter is dust-free.
   5. If heating is required, make sure the extruder is placed inside the glass container (use DI water) and placed on hot plate.  Begin extruding until the water is at the correct temperature.
   6. Aliquot the 9 ml of lipids in cryo-vials (1 ml each).
   7. Do 10 freeze-thaw cycles with liquid nitrogen and warm tap water.  Be very careful when handling liquid nitrogen -  use face mask and cryogenic gloves.
   8. Assemble extruder as shown on manual and extrude each 1 ml vial 10 times.  Remember drain disk goes first, then 2 membrane filters shiny side up.
   9. After all 9 samples are extruded, prepare 1 L of 1X PBS or the buffer utilized to prepare the liposomes.
   10. Size liposomes using ZetaPALS analyzer.
   11. Soak a dialysis cassette (MWCO of 20,000 Da) for a couple of minutes in the 1L buffer.
   12. Using an 18 gauge needle (pink) and a plastic syringe, inject very slowly and carefully the 9 ml of extruded liposomes.
   13. Attach a buoy or floatie onto the cassette and dialyze overnight by placing on a hot/stir plate (no heat) at 400 RPM.  Make sure to cover with foil.

Day 3

    1. Extract sample from dialysis cassette using a needle again (use a different port than the one you used to inject sample in).  Transfer to a 15 ml falcon tube covered in foil and store at 4C.
[[Category:Protocols]]