Murine Cortical Neuron Culture Protocols

Primary cortical neuronal cultures were prepared from embryonic day 16 C57Bl/6 mice as described previously.^{60 (link)} Primary cortical neurons were grown in a culture medium composed of Neurobasal medium (Invitrogen, Carlsbad, CA, USA), 2% B27 supplement (Invitrogen), 2 mM l-glutamine, and 1% penicillin-streptomycin, as described previously.^{60 (link)} After 3 days of culture, a third of the medium was replaced with fresh, L-glutamine-free medium containing 5 μM cytosine arabinofuranoside (Sigma) to arrest non-neuronal cell growth. Experiments were conducted on the 12th day of culture, by which time the cultures consisted primarily of neurons (>95% MAP-2–immunoreactive cells; MAP-2 was obtained from Chemicon (Temecula, CA, USA).