Isolation and Culture of Mouse Hippocampal Neurons

Primary neurons were isolated from the hippocampi of both male and female C57Bl6/N mouse pups at post‐natal day 0 or 1. For the isolation and culture of hippocampal neurons, we followed the protocol of Beaudoin et al (2012 (link)) with slight modifications. Briefly, hippocampi were extracted into Hibernate A media (Gibco) and incubated at 37°C with papain solution for 20 min. Papain solution was removed, and trypsin inhibitor was added for 5 min. Hippocampi were then washed 3 times in pre‐warmed plating media (Neurobasal A, B27 supplement, GlutaMAX, Horse serum, 1 M HEPES pH 7.5) before being triturated 8–10 times. The suspension was strained, and 800,000 cells were seeded onto 6‐well plates coated with poly‐L‐lysine. Media was changed to neuron media (Neurobasal A, B27 supplement, GlutaMAX, Penicillin/Streptomycin) 4 h post‐seeding. Primary neurons were maintained at 37°C, 5% CO₂ or as indicated at 32°C. 5‐fluoro‐2′‐deoxyuridine (Sigma‐Aldrich) was added at a final concentration of 7.15 μg/ml to inhibit glial growth (Vossel et al, 2015 (link)). A third of the media was changed for fresh media every 4–5 days. Experimental procedures lasting 24–48 h were started at day 19–20 D.I.V. to finish at 21 D.I.V.