Using the recommended kit, proceed to prepare 95 microliters of the nucleofection solution in a 1.5 milliliter tube for each desired nucleofection condition. Also prepare a T25 flask filled with four milliliters of pre-warmed complete medium per condition and keep it in the incubator until use. To perform nucleofection, centrifuge the desired number of cells for five minutes at 300 G before removing the supernatant.
Add one milliliter of DPBS and re-suspend the pellet homogenously by pipetting up and down 10 times. After repeating the centrifugation, re-suspend the pellet in 95 microliters of nucleofection solution. Combine the 95 microliters of cells with a pre-mixed solution containing five microliters of each plasmid selected for nucleofection.
After transferring this solution to a cuvette, place it in the nucleofector device to nucleofect the cells with the plasmids using the optimized neural stem cell program of the nucleofector system. After completing the electroporation, using the Pasteur pipette provided in the kit, carefully introduce warm complete medium into the cuvette. Transfer the contents of the cuvette to the previously prepared T25 flask containing pre-warmed complete medium.
Incubate the nucleofected cells in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide for three to five days and visualize the nucleofected neurospheres.
