Isolation and Culture of Cortical Neurons from Mice

Twenty-four hours after the mechanical trauma, cortical neurons were isolated from P5-6 mice and plated on top of the reactive astrocytes as previously reported13 (link)14 (link). Cortices were dissected in Hibernate medium (Life Technologies) and incubated for 30 min at 30 °C with 2 mg/ml papain (Sigma-Aldrich, Australia). After enzymatic digestion, cells were dissociated by gentle pipetting and the cellular suspension was purified by Optiprep (Sigma-Aldrich) gradient to isolate neurons from the other cells as previously reported13 (link)14 (link). Cortical neurons thus dissociated were plated at a density of 20,000/well on the top of pre-prepared astrocyte cultures.