Technical reference guide, experimental set up as described in the amaxa protocol for rat hippocampal neurons for the standard Nucleofector (O-003 or O-005) and for the shuttle (96-CU-133). Critical comment:\"Transfection efficiencies of 95% can, for example, be achieved with the pmaxGFP plasmid, which is included as a control with nucleofector kits. By contrast, in our experience, the transfection efficiencies obtainable with the 96-well shuttle for primary neurons tend to be lower than that with the nucleofector device and generally range between 30% and 50%. But amaxa continously improve the protocols\". \"Remarkable, no influence on morphology and functionality of the cells\".
Transfection of foreign DNA is widely used to study gene function. However, despite the development of numerous methods, the transfer of DNA into postmitotic cells, such as neurons, remains unsatisfactory with regard to either transfection efficiency or cytotoxicity. Nucleofection overcomes these limitations. Direct electroporation of expression plasmids or oligonucleotides into the nucleus ensures both good cell viability and consistently high transfection rates. This allows biochemical analyses of transfected neurons, for example, western blot analyses of protein levels after RNA interference (RNAi) knockdown or microRNA transfection. We provide comprehensive protocols for performing nucleofection with high efficiency on primary neurons. The focus is on the recently developed 96-well shuttle system, which allows the simultaneous testing of up to 96 different plasmids or experimental conditions. Using this system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression. The protocol typically takes between 2 and 3 h.