Primary Cortical Neuron Culture Protocol

Primary cortical neurons were prepared as described before [52 (link)]. Briefly, cortices from mice embryos at embryonic day 16.5 were dissociated mechanically, and neurons were resuspended in Neurobasal™ medium supplemented with B27 (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) containing 30 U/mL Penicillin, 30 mg/mL Streptomycin (Sigma-Aldrich, Milan, Italy), 0.75 mM Glutamax (Gibco™, Thermo Fisher Scientific), and 0.75 mM L-Glutammine (Gibco™, Thermo Fisher Scientific). Depending on the experiment, neurons were seeded 80.000 cells/1.9 cm² on 0.1 mg/mL Poly-D-Lysine-coated glass coverslip (Sigma-Aldrich, for transfection with miRNA expression vectors) or 300.000 cells/10 cm² on 0.02 mg/mL Poly-D-Lysine-coated 6-multiwell plates (for transfection with miRNA mimics) and maintained at 37 °C under a 5% CO₂ humid atmosphere. Three days after seeding, half of the medium was replaced with 24 h astrocyte-conditioned medium. Then, half of the medium was changed every seven days for up to a maximum of four weeks.