Deoxyribonulease 1

Neurons were prepared from the cerebral cortices of mice fetuses (postnatal day 0) based on a modification of a previously described procedure (Kakizawa et al., 2012 (link), Kanemaru et al., 2007 (link)). Briefly, minced cerebral cortices were treated with 1.0% (w/v) trypsin and 0.1% (w/v) Deoxyribonulease I (Sigma-Aldrich) in Ca^2 +/Mg^2 +-free phosphate-buffered saline (PBS) (Takara, Shiga, Japan) for 5 min at room temperature (RT). Cells were washed with Neurobasal-A medium supplemented with 5% (v/v) fetal bovine serum (FBS), penicillin (100 units mL^− 1), streptomycin (100 units mL^− 1), B-27 supplement, and 2 mM l-glutamine (Gibco, ThermoFisher Scientific, Grand Island, NY, USA) and dissociated by triturating with a fire-polished Pasteur pipette in Ca^2 +/Mg^2 +-free PBS containing 0.05% (w/v) Deoxyribonulease I and 0.03% (w/v) trypsin inhibitor (Sigma-Aldrich). Dispersed cells were plated at 1.0 × 10⁵ cells cm^− 2 on glass slide coated with poly-l-lysine and laminin (Sigma-Aldrich). Cells were then cultured at 37 °C under a humidified atmosphere containing 5% CO₂. The medium was changed every 2 d by replacing half of the old medium with fresh FBS-free medium. Cells cultured for 7–10 d were used for experiments.