Primary neuron culture and transfection

Primary hippocampal neuron isolations were performed from E18.5 mouse embryos. Briefly, hippocampi were collected into ice-cold 5 ml of 1x HBSS buffer containing 10% sucrose, then 1 ml of 0.25% trypsin was added for digestion at 37 °C for 15 min. The tissues were gently triturated with a glass Pasteur pipette to isolate the cells. 6 × 10⁴ disassociated cells per 24-well plate were seeded in MEM medium (Thermo Fisher) supplemented with 1% glucose, 1x sodium pyruvate, 1% penicillin/streptomycin, 10 mM HEPES (Thermo Fisher), 2% B27 (Thermo Fisher), 1% FCS and 1 mM L-glutamine. The neurons were recovered for 1 to 3 h in a cell culture incubator at 37 °C in 5% CO₂ and 20% O₂. Then, the plating medium was replaced with Neurobasal Medium (Thermo Fisher) supplemented with 1% penicillin/streptomycin, 10 mM HEPES, 2% B27 and 0.5% GlutaMAX (Thermo Fisher). The neuron medium was refreshed every 3 days.

For transfection, neurons were cultured for 7 days and transfected with Lipofectamine 2000. Briefly, 15 ml of Neurobasal Medium (NBM) supplemented with 37.5 µl of 200 mM L-glutamine was used as incubation medium. Separately, 0.9 µg DNA and 1.65 µl Lipofectamine 2000 were diluted in 50 µl NBM per well of 24-well plate, mixed and incubated for 30 min at RT. The conditioned medium of the neurons was transferred to a new 24-well plate and kept in the 37 °C incubator. 500 µl of incubation medium and 100 µl of DNA + Lipofectamine mixture were added to the neurons and incubated at 37 °C for 45 min. The transfection mixture was then replaced with the conditioned medium. Transfected neurons were fixed with 4% PFA/sucrose and subjected to immunofluorescence staining 7 to 10 days after transfection.