Isolation of Primary Cerebral Neurons

Primary cultures of cerebral neurons were obtained as described previously (Hilgenberg and Smith, 2007 (link)). In brief, mouse foetuses (embryonic day 17 [E17]) were removed from the uterus, and the individual foetuses were freed from the embryonic sacs. The brain and cortical tissue were dissected and placed in high-glucose Dulbecco’s modified Eagle’s medium (DMEM-HG) without phenol red (Gibco, Grand Island, NY, USA; Cat. No. 31053028). Papain solution (10 U/mL; Sigma‒Aldrich, St. Louis, MO, USA; Cat. No. LS003126) was added to these cerebral tissues, which were then incubated for 15 min at 37°C in a 5% CO₂ incubator. Dissociated cortical cells were plated on poly-L-lysine (Sigma‒Aldrich; Cat. No. P4832)-coated cell culture dishes and cultured in DMEM-HG (Gibco, Grand Island, NY, USA; Cat. No. 31053028) containing 10% foetal bovine serum (Gibco, Australia; Cat. No. 10099141) and 1% penicillin/streptomycin (P/S; Invitrogen, Carlsbad, CA, USA; Cat. No. 15140148) at a density of 1.0×10⁶ cells/mL. Four hours after seeding, the medium was replaced with neurobasal medium (NM; Gibco, Carlsbad; Cat. No. 21103049) supplemented with B-27 (Gibco, Grand Island, NY, USA; Cat. No. 17504044). Cells were cultured in a humidified incubator at 37°C with 5% CO₂. The medium was changed every 3 days. Cultures were used for in vitro experiments after 7 days.