B27 serum free

Primary hippocampal neurons were prepared from rats and mice as previously described (Farías et al., 2016 (link)). In brief, embryonic day 18 (E18) rats or mice were harvested and euthanized by decapitation. The brain was removed from the skull, and hippocampi were dissected in fresh Hank’s medium and treated with 0.25% trypsin (GIBCO) for 15 min at 37°C. The tissue was then washed twice with Hank’s medium and resuspended in plating medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) without phenol red, supplemented with 4.5 g/L glucose, 25 mM HEPES, 10% heat-inactivated horse serum (GIBCO), 100 U/mL penicillin and 100 μg/mL streptomycin. The hippocampi were then disrupted mechanically with a glass Pasteur pipet whose tip was narrowed to around 50% of its original diameter. The cells were counted, and 80,000 cells plated on 18-mm glass coverslips previously coated with polylysine (Sigma) and 5 μg/mL laminin (Roche). After 4 h, the medium was changed to complete Neurobasal medium (CNB) consisting of Neurobasal medium (GIBCO), supplemented with 1X B27 serum free (Thermo Scientific), 4.5 g/L glucose, and 100 U/mL penicillin-streptomycin (GIBCO).