This article details the electroporation method for delivering plasmid DNA into primary neuronal cells. The process involves preparing a cell suspension, applying electrical pulses, and allowing DNA translocation into the nucleus.
For electroporation-based plasmid DNA delivery into primary neuronal cells, begin with a suspension of cells in suitable electroporation media. Add fluorescent protein-encoding plasmid DNA to prepare the electroporation mixture.
Gently transfer the electroporation mixture into an electroporation cuvette, avoiding air bubbles that could negatively affect electroporation efficiency. Transfer the cuvette to the electroporator's cuvette holder; set the electroporation parameters. Initiate electroporation.
The applied high-voltage short electrical pulses across the cell membrane cause differential charge accumulation on either sides of the membrane, increasing the transmembrane potential, the voltage across the cell membrane.
When the induced transmembrane potential exceeds the lipid bilayer breakdown potential, the lipid bilayer destabilizes with the localized configuration change, forming transient hydrophilic pores on the cell membrane. The pores with adequate size provide a pathway for plasmid DNA to enter the cell cytoplasm.
On completion of electroporation, the lipid bilayer reseals, restoring the cell membrane bilayer integrity, thereby entrapping the plasmid DNA. Further, during the phase of cell cycle when the nuclear membrane, the double-layered membrane surrounding the nucleus, disassembles, plasmid DNA translocates to the nucleus.
Plate electroporated cells into media-containing wells of a multi-well plate with protein-coated coverslips. Incubate to allow cells to adhere to the protein-coated coverslip. Successfully transfected cells containing plasmid DNA express the fluorescent proteins.
To begin, add 0.5 milliliters of culture medium into each wall of the 24-well culture plate containing coated coverslips, and keep it warm at 37 degrees Celsius in a carbon dioxide incubator. Pipette the required number of cells into a sterile 1.5-milliliter microcentrifuge tube and spin at 200 x g for 5 minutes at room temperature.
Discard the supernatant, and resuspend the cell pellet in 200 microliters of Opti-MEM. Repeat this procedure twice to ensure no residual culture medium is present in the tube. Set the parameters of electroporation as listed. Pipette the electroporation reaction gently to mix well, and use a long P200 pipette tip to transfer an exact volume of 100 microliters of the mixture into the two-millimeter gap cuvette.
Once the cuvette is inside the cuvette chamber, press the omega button of the electroporator, and note the impedance value by adhering to a precise volume of 100 microliters. The range of impedance values should be approximately 30 and 35.
Press the Start button to initiate the pulse. Record the measured current values and jewels shown on the reading frame. Remove the cuvette from the chamber.
Immediately add 100 microliters of pre-warmed culture medium into the cuvette, and resuspend it by pipetting up and down two to three times. Immediately transfer the cell suspension to the 24-well plate previously prepared. Incubate the cells at 37 degrees Celsius in a carbon dioxide incubator.
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