Neuron culture medium

Dissociated cerebral cortical neurons were prepared from embryonic day 17.5 (E17.5) individual littermates. Neurons were plated at a density of 6.4 × 10⁴ cells/cm² onto poly-D-lysine-coated coverslips of glass bottom culture dishes (MatTek, Ashland, MA) in Neurobasal-A medium (Thermo Fisher Scientific, Waltham, MA) containing B-27 supplement (Thermo Fisher Scientific), 0.5 mM glutamine and 25% Neuron culture medium (Sumitomo Bakelite, Tokyo, Japan). Cultures were maintained at 37 °C in a 5% CO₂ incubator.

Neurons were prepared from the VTA and striatum of C57BL/6J mouse embryos (E16). After removing the cerebrum, the VTA and striatum were dissociated into single-cell suspensions with a cell strainer (100 μM pore size, BD Biosciences, San Jose, CA, USA). The cells were seeded on poly-L-lysine coated coverslips and plated at a density of 2.5 × 10⁴ cells per cm² in neuron culture medium (Sumitomo Bakelite, Tokyo, Japan) with 5 μM Ara-C (Sigma-Aldrich, St. Louis, MO, USA) and 1× antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA). The cells were incubated in the culture medium in a humidified incubator at 37 °C and 5% CO₂.

Primary neuronal cultures were prepared from the cortices of C57BL/6 mouse embryos at embryonic Day 17 as described previously [20] (link). Briefly, cortical fragments were dissociated into single cells in dissociation solution (Sumitomo Bakelite, Akita, Japan), and resuspended in neuron culture medium (Sumitomo Bakelite). Neurons were seeded onto 12-mm polyethylenimine-coated glass coverslips (Asahi Techno Glass Corp., Chiba, Japan) at a density of 5.0×10⁴ cells/well in 24-well culture plates and were incubated at 37°C in a humidified atmosphere containing 5% CO₂. The purity of the cultures was >95% as determined by NeuN-specific immunostaining. Mouse brain capillary endothelial cell line MBEC4 [21] (link) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Confluent monolayer of MBEC4 cells was used as an established BBB model as described previously [22] (link).