After the freeze-thawing cycles are completed, the vesicle suspension is transferred to one of the syringes of the extrusion kit (Figure ). Small amounts of NaNO3 solution (a few drops) are used to rinse the remaining vesicles from the flask and are added to the rest of the solution in the syringe, which is then filled up to 1 mL with the NaNO3 solution. The mixture is then passed 29 times through the membrane to produce monodisperse large unilamellar vesicles. Excessive pressure on the pistons should be avoided. The extrusion cycles are ended on the opposite side of the membrane, so that the solution has effectively been filtered.