Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells

1. Electroporation for Hh receptor transgene overexpression:  pEGFP-Smo

1. For electroporation using a 2 mm gap cuvette, prepare the following plasmid-cell electroporation mixture for each reaction of electroporation: 1.2 × 10⁶ mouse primary cerebellar granule cell precursors cells and 10 µg of pEGFP-mSmo (adjust the DNA stock concentration to ~2-5 µg/µL in Tris-EDTA buffer or sterile dH₂O) in 100 µL of Opti-MEM. 
   NOTE: Reduce the total cell number if the cells are insufficient (though this may reduce the electroporation efficiency). However, do not adjust the amount of plasmid nor the total volume of Opti-MEM used per cuvette. If the total cell number required for the experiment exceeds 1.5 × 10⁶ cells, scale up the electroporation mixture accordingly and perform multiple electroporation reactions separately. The cuvette may be reused up to 5 times.
2. Prepare the post-electroporation cell seeding plate by adding 0.5 mL of culture medium into each well of the 24-well culture plate containing coated coverslips and keep it warm at 37 °C in a CO₂ incubator. To prepare the coverslips for cell attachment, incubate autoclaved 12 mm glass coverslips with 100 µg/mL of poly-D-lysine (PDL, 1 mg/mL in sterile dH₂O) for at least 1 h at 37 °C. Keep the coverslips in the same PDL solution at 4 °C until use.
3. Pipette the required number of cells into a sterile 1.5 mL microcentrifuge tube and spin at 200 × g for 5 min at RT. Discard the supernatant and resuspend the cell pellet in 200 µL of Opti-MEM. Repeat this step twice with Opti-MEM to ensure no residual culture medium is present in the tube.  
   NOTE: For every well/coverslip of a 24-well plate, electroporate and seed 1.2-1.3 × 10⁶ cells/well to obtain ~70-75% cell confluency on the next day of culture.
4. Set the parameters of electroporation as shown in Table 1.
5. Pipette the electroporation reaction gently to mix well and use a long P200 tip to transfer an exact volume of 100 µL of the mixture into the 2 mm gap cuvette. Avoid forming bubbles.
6. Place the cuvette in the cuvette chamber.
7. Press the Ω button of the electroporator (see the Table of Materials) and make a note of the impedance value, which should be ~30-35. To ensure the electric impedance value Ω falls within the range of 30-35, adhere to a precise volume of 100 µL.
8. Press the start button to initiate the pulse.
9. Record the values of the measured current and joules shown on the reading frame.
10. Remove the cuvette from the cuvette chamber.
11. Immediately add 100 µL of prewarmed culture medium into the cuvette and resuspend it by gentle pipetting up and down 2-3 times. Immediately transfer the cell suspension to the 24-well plate containing coated coverslips prepared in step 1.2.
    NOTE: To minimize cell death, seed the cells immediately after electroporation.
12. Incubate the cells at 37 °C in a CO₂ incubator. Leave the cells undisturbed for 3 h to avoid cell detachment.
13. At 3 h post seeding, gently aspirate and discard half of the supernatant medium to remove floating dead cells and debris and replace with the same amount of prewarmed culture medium.
14. Incubate the cells at 37 °C in the CO₂ incubator.

Table 1: The electroporation parameters of mouse primary GCPs and primary neurons using Super Electroporator NEPA21 TYPE