UA Biophysics:Protocols: GUV

About the lipids

1. In a glass container, mix te lipids in chloroform at a concentration of 0.20 mg/ml (0.2 mM).
2. Deposit 10 µl of lipid mix in 2 µl drops at the tip of each pair of Pt-electrodes.
3. Dry the solvent under a stream of nitrogen gas.
4. Remove the remaining traces by lyophilizing the electrodes with their lipid deposits for 1 hour.

Another option:

1. Prepare LUVs at 0.20 mg/ml
2. Deposit 10 µl of lipid mix in 2 µl drops at the tip of each pair of Pt-electrodes.
3. Dry the water under a stream of nitrogen gas.
4. Remove the remaining traces by lyophilizing the electrodes with their lipid deposits for almost 3 hour.

Electroformation protocol

1. Prepare 50 ml of 200 mM sucrose solution and 50 ml of 200 mM glucose solution.
2. Add 1 ml of sucrose solution in each electroformation cell. There is one cell for each pair of electrodes.
3. Immerse the electrodes in their electroformation cell.
4. Apply to each electrode a sine wave of peak-to-peak voltage of 0.25 V and frequency of 10 Hz for 5 minutes.
5. Increase the voltage (Vpp) by 0.25 V every 5 minutes until reaching a maximum peak-topeak voltage of 3.5 V . The frequency remains constant at 10 Hz. During this step,GUVs gradually start to form from the deposits on the electrodes.
6. Set the voltage at 3.5 Vpp for 1 hour, the frequency remains constant at 10 Hz. During this step, GUVs increase progressively in size.
7. While the voltage remains constant at 3.5 Vpp, decrease the frequency by 0.5 Hz every 5 minutes until reaching a minimum frequency of 4 Hz. The frequency is reduced to promote vesicle closure and future detachment from their breeding electrodes.
8. Gently tap the electroformation cells to liberate the GUVs from the electrodes.

GUV observation

1. Make a circle of vacuum grease on a microscope slide.
2. Add 100 µl of glucose solution and 100 µl of GUVs solution inside the circle.
3. Put a coverslip on the microscope slide.
4. Carefully place the sample under the microscope.

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