Unilamellar Liposomes: Extrusion

Resuspend the lipid film in 150 μl of 20 mM Hepes pH 7.0 and vortex for 1 min. The solution should be cloudy (see Note 3).

Let the liposome suspension stand for 1 h at room temperature (RT) to hydrate.

Disrupt the nonuniform, multilamellar liposomes by ten cycles of freeze-thaw between an acetone-dry ice bath and a water bath set above the transition temperature (for rapid thawing we use a water bath set to 50 °C).

Assemble the extruder according to the instructions on the website of Avanti Polar Lipids, Inc. Briefly:

In the retainer nut (side B), place the Teflon bearing followed by the internal membrane support, O-ring side up.

Inside of the O-ring, place a filter support.

Place a polycarbonate membrane with the desired pore size over the filter support such that it covers the internal membrane support O-ring completely. We use membranes with a pore size of 100 nm for our experiments.

Place a second filter support in the center of the polycarbonate membrane (see Note 4).

Place the second internal membrane support on top of the stack, O-ring side down.

Slide the extruder outer casing (side A) over the top internal membrane support and screw it into the retainer nut. Tighten by hand, making sure that the apexes of the outer casing and retainer nut line up.

Rinse the extruder syringes three times with water and three times with 20 mM Hepes pH 7.0.

To reduce the dead volume in the extruder, pass one to three syringe volumes worth of 20 mM Hepes pH 7.0 through the extruder and discard.

Take up the liposome solution into syringe A and insert it into side A of the extruder set up. Remove any excess buffer that is pushed out of side B with a Kim wipe.

Insert an empty syringe B into side B of the extruder setup.

Place the assembly in the holder (see Note 5).

Gently push the liposome solution from syringe A to syringe B and back again, repeating 10–60 times.

Push the liposome solution into syringe B and remove the assembly from the holder. Holding the assembly vertically with syringe B on the bottom, remove syringe A, and pull down on the plunger in syringe B to remove all of the liposome solution from the chamber. Transfer the extruded liposome solution from syringe B into an Eppendorf tube and store at RT (or above the transition temperature) until use (see Note 6).

Clean the extruder syringes three times with 20 mM Hepes pH 7.0, three times with water, and once with 100 % ethanol (see Note 7).

Disassemble the extruder and discard the membrane and filter supports. Rinse all of the components with milli-Q water.