Version 0.1.0, 14 August 2018
Contributors: Aaron Engelhart (University of Minnesota), Jan Gregrowicz (Caltech), Joseph Heili (University of Minnesota), Zoila Jurado (Caltech), Neha Kama (Northwestern), Akshay Maheshwari (Stanford), Richard Murray (Caltech), Milena Popovic (Blue Marble Space), Pasquale Stano (University of Salento), Kazhito Tabata (University of Tokyo), Paola Torre (University of Pennsylvania).
This protocol prepares phospholipid films with and without a lissamine rhodamine membrane dye to be hydrated later to prepare lipid vesicles. This procedure works best for studies with bulk small unilamellar vesicles, but can be used to prepare giant (>1 um) vesicles (just skip the extrusion step).
A. Glass vials (any will do, but we use 2mL Fisherbrand Class B Clear Glass Threaded vials: cat #: 03-339-21A
B. Glass syringes of various sizes (ex. Hamilton gastight cat #: 14-815-238, but any will do) for use with lipids and chloroform
C. Phosphate buffered saline 290 mOsm (Sigma, P4417-100TAB)
D. Mini Extruder (Avanti Polar Lipids)
Final reaction conditions:
| Reagent | Source | Final working concentration |
|---|---|---|
| DOPC | Avanti 850375C | 2E-5 mols/vial (1 mL of hydration yields 20 mM vesicles)* |
| Rhodamine Lissamine PE | Avanti 810150 C | 0.1 mol % of POPC |
| Cholesterol | Santa Cruz Biotech sc-202539 | 2X molarity of DOPC, if used |
* For microscopy, lower concentration of vesicles hydrated in low salt solutions yields higher quality vesicles for imaging (recommend you make 125 uM vesicles once hydrated)
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Determine the volume and molarity DOPC vesicles you want to create, e.g. 1 mL of 20 mM DOPC vesicles = 2E-5 mols of DOPC. The stock of DOPC is 25 mg/mL. To make giant, nice looking vesicles, it is best to prepare a more dilute sample of vesicles (like 200 uM). For small unilamellar vesicles, you can work with much higher concentrations of lipids (we can go up to 130 mM for DOPC)
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Determine the dye and molar % of the dye : DOPC you would like ex. for 0.1 mol% rhodamine DHPE :
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2.5e-6 moles DOPC×(0.1 moles Rhod Liss PE)/(100 moles DOPC)×L/(moles Rhod in stock)=volume Rhod Liss PE stock to add
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Add 2e-5 mols DOPC and appropriate amount of Rhodamine PE. Transfer with a Hamilton (glass) syringe into a glass vial since you are working with organic solvents. Cap and vortex briefly to ensure the two components are well mixed.
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Remove cap and allow the chloroform to evaporate away in the hood.
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Place the vial into a vacuum chamber for > 1 hrs or leave overnight in order to remove all residual traces of solvent. Removal of residual chloroform from the lipid film is very important for high quality vesicles.
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Cap and store vials in freezer until ready to be used and hydrate with 1 mL of aqueous hydration buffer when you are ready to prepare vesicles.
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Add 1 mL of PBS to each vial, place in 60 degC oven for 1 hr, then vortex 10 seconds. Lipids should easily assemble. If clumps or aggregates persist after several vortexing attempts, this is often a sign something went wrong in vesicle assembly. The heating step is not necessary and can be excluded, but helps when other membrane components are involved like diblock copolymers.
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Extrude films at room temperature through 100 nm polycarbonate membranes using a mini extruder and heating block described on the Avanti website, following the procedure described here (7 passes through membrane is fine, 9-11 is better, more than that is unnecessary according to my post extrusion analysis of vesicle size distribution on DLS.) Always perform an odd number of passes through the membrane so no vesicles that have never passed through the membrane end up in the final sample.
- Note: additional information on this step is available in section 3 of step 7 in the JoVE protocol on fatty acid-containing liposomes.
- Set up columns (e.g. Bio-Rad Poly-Prep chromatography columns) containing 6 mLs of Sepharose 4B (size exclusion column media). Purification should be conducted on the same day as a subsequent assay.