B27 serum free supplement for neural cell culture

Hippocampal neurons were isolated from the forebrains of 17-day-old rat embryos, as previously described [33 (link)]. Briefly, the neurons were seeded at a density of 5 × 10⁶ cells/cm² in poly-D-lysine-coated culture dishes and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) containing 10% (v/v) fetal bovine serum (FBS, Thermo Fisher Scientific Inc., USA). After 30 min, the culture medium was changed to Neurobasal Medium (NeuB, Thermo Fisher Scientific Inc., USA) with B27 serum-free supplement for neural cell culture (Thermo Fisher Scientific Inc., USA), 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2.5 mg/mL Fungizone (Thermo Fisher Scientific Inc., USA). The cell cultures were incubated at 5% CO₂ in a humidified environment at 37 °C. For all experiments, the neuronal cells were used after 14 days of in vitro (DIV) culture. Cytosine arabinoside was used to prevent non-neuronal cell contamination.

Hippocampal neurons isolated from the forebrains of 17-day-old rat embryos, as described in (Arrazola et al. 2009 (link)), were seeded in poly-D-lysine-coated culture dishes at a density of 5×10⁶ cells/cm² and cultured in Dulbecco’s modified Eagle’s medium (cat: D5030, DMEM, Invitrogen, USA) containing 10 % (v/v) fetal bovine serum (cat: 16000044, FBS, Thermo Fisher Scientific Inc., USA). After 30 min, the culture medium was changed to Neurobasal medium (cat: 21103049, NeuB, Thermo Fisher Scientific Inc., USA) supplemented with B27 serum-free supplement for neural cell culture (cat: 15504044, Thermo Fisher Scientific Inc., USA), 2 mM L- glutamine (cat: 21051024, Thermo Fisher Scientific Inc., USA), 100 U/mL penicillin plus 100 mg/mL streptomycin (cat: 15070063, Thermo Fisher Scientific Inc., USA), and 2.5 mg/mL fungizone (cat: 15240112, Thermo Fisher Scientific Inc., USA),. Cells were incubated in 5 % CO₂ in a humidified environment at 37 °C. Neuronal cells were used after 14 days of in vitro culture (DIV 14) in all experiments. Cytosine arabinoside (cat: C1768, Ara-C, Sigma-Aldrich, USA) was used to prevent non-neuronal cell contamination.