Micropipette tips

For electroformation with Platinum (Pt) electrodes, the wires can be placed vertically or horizontally.^{35 ,36 (link),38 (link)} Here, we utilize the horizontal configuration in order to monitor the vesicles during the electroformation process using a microscope. A home built device consisting of a PVC (polyvinyl chloride) chamber to house the Pt wires was used. The Pt wires are removable from the PVC chamber for cleaning purposes. Coverslips are attached at the bottom of chamber using vacuum grease. Slowly 5-10 μl of lipid solution is spread on both sides of the Pt wires. The device is placed in a dessicator for 3 hours to evaporate all solvents. The chamber is slowly filled with 2 ml of 500 mM sucrose in 0.3 mM of NaCl solution and the Pt wires are directly connected to signal generator (Agilent Technology, USA) for 2 hours at 50 Hz and voltage 1.5 V_pp. The vesicles are smoothly aspirated using 1 ml micropipette tips (Eppendorf, Germany). The GUV harvest is diluted for analysis in a similar way like the spontaneous swelling method.

The glass slides (50 mm × 50 mm ITO slides with 50 Ohms resistance, Delta technologies, USA) are cleaned with acetone and isopropanol and triple rinsed with bi-distilled water. The stock solution of POPC in choloroform is diluted to 6 mM from which 7-10 μl of the solution is spread on the conductive sides of each slides using gas-tight glass syringe (Hamilton, USA). The lipid-coated slides are stored in dessicator for 3 hours to evaporate all organic solvents. Then the slides with facing conductive sides are assembled to sandwich a 2 mm thick Teflon spacer and are clipped together. Through a hole in the Teflon spacer, the chamber is gently filled with 2 ml of 500 mM sucrose solution in 0.3 mM of NaCl to avoid film disruption. Next, the conductive side of ITO is connected to a signal generator (Agilent Technology, USA) for 2 hours at 50 Hz and voltage 1.5 V_pp using copper tapes (3M, USA). After electroformation, the vesicles are smoothly aspirated using 1 ml micropipette tips (Eppendorf, Germany). The GUV harvest is diluted for analysis in a similar way like the spontaneous swelling method.