For Lower cell number, 5E+5 neurosphere-derived NSCs were resuspended in 20µl P3 nucleofection solution containing 0.5 µg plasmid DNA. Cells were transferred into the nucleofector cuvette. After the pulse application, 180 µl prewarmed neurosphere medium was added to the electroporated NSCs in the cuvette. NSCs were gently resuspended in the cuvette and transferred into a sterile 1.5 ml tube. After centrifugation with 80×g for 5 min at room temperature, the supernatant was discarded and the cell pellet was resuspended in 500 µl neurosphere medium containing EGF and FGF2 (20 ng/ml).
For higher cell numbers, 2.5–5E+6 neurosphere-derived NSCs were resuspended in 100 µl P3 nucleofection solution with 5 µg plasmid DNA and then nucleofected in the 100µl nucleofection cuvette. The transfected NSCs were resuspended in 500 µl prewarmed neurosphere medium, transferred to a 15 ml Falcon tube and centrifuged also at 80×g for 5 min at room temperature. The supernatant was discarded and the pellet was resuspended in neurosphere medium containing the same growth factors as above and cultivated as neurospheres over night at 37 °C.