35. Remove HBSS 1Â from the transwell inserts and plate 1 Â 10
6
cells per insert in 500 μL of Neuronal Culture Media-Plating
(see Note 3).
36. Keep the neurons in humidified incubator for at least 7 days
before performing NEP. On day 4 replace one-third of
media with fresh Neuronal Culture Media prepared without
glutamate (see Notes 4 and 5).
3.3 NanochannelBased Electroporation
(NEP)
1. Set up the NEP platform by placing the PDMS reservoir gasket
on top of the gold sleeve.
2. Connect the negative electrode of the power source to the gold
sleeve and the positive electrode to the gold-plated electrode.
3. Dilute 25 μg of plasmid DNA into 500 μL of PBS (final
concentration 0.05 μg/μL).
4. Transfer 350 μL plasmid DNA solution or PBS as control to
the PDMS reservoir gasket.
5. Remove and save the Neuronal Culture Media from the neurons, add 1 mL of PBS to the transwell insert.
6. Place the transwell insert on top of the PDMS reservoir gasket,
avoiding the formation of bubbles in the plasmid DNA solution underneath.
7. Place the gold-plated electrode with plastic casing previously
connected to the positive electrode inside the PBS solution of
the transwell inserts at a distance of approx. 6 mm from the
surface where the cells are cultured.
8. Using the power source deliver a series of square electric pulses
with the following settings:
Voltage: 275 mV.
Pulse Length: 35 ms.
Number of pulses: 10.
Interval between pulses: 0.1 s.
9. After the pulses are done, remove the gold tip and discard the
PBS inside the transwell insert.
10. Return the transwell insert to the multiwell plate and add the
Neuronal Culture Media previously saved.
11. Incubate from 16 to 24 h and check the expression of your
gene of interest.
4 Notes
1. If is too difficult to get a single cell suspension during the
trituration step, check the concentration of trypsin and/or
DNase I. If necessary, replace the batches of both trypsin and
150
Diego Alzate-Correa et al.
6
cells per insert in 500 μL of Neuronal Culture Media-Plating
(see Note 3).
36. Keep the neurons in humidified incubator for at least 7 days
before performing NEP. On day 4 replace one-third of
media with fresh Neuronal Culture Media prepared without
glutamate (see Notes 4 and 5).
3.3 NanochannelBased Electroporation
(NEP)
1. Set up the NEP platform by placing the PDMS reservoir gasket
on top of the gold sleeve.
2. Connect the negative electrode of the power source to the gold
sleeve and the positive electrode to the gold-plated electrode.
3. Dilute 25 μg of plasmid DNA into 500 μL of PBS (final
concentration 0.05 μg/μL).
4. Transfer 350 μL plasmid DNA solution or PBS as control to
the PDMS reservoir gasket.
5. Remove and save the Neuronal Culture Media from the neurons, add 1 mL of PBS to the transwell insert.
6. Place the transwell insert on top of the PDMS reservoir gasket,
avoiding the formation of bubbles in the plasmid DNA solution underneath.
7. Place the gold-plated electrode with plastic casing previously
connected to the positive electrode inside the PBS solution of
the transwell inserts at a distance of approx. 6 mm from the
surface where the cells are cultured.
8. Using the power source deliver a series of square electric pulses
with the following settings:
Voltage: 275 mV.
Pulse Length: 35 ms.
Number of pulses: 10.
Interval between pulses: 0.1 s.
9. After the pulses are done, remove the gold tip and discard the
PBS inside the transwell insert.
10. Return the transwell insert to the multiwell plate and add the
Neuronal Culture Media previously saved.
11. Incubate from 16 to 24 h and check the expression of your
gene of interest.
4 Notes
1. If is too difficult to get a single cell suspension during the
trituration step, check the concentration of trypsin and/or
DNase I. If necessary, replace the batches of both trypsin and
150
Diego Alzate-Correa et al.