Astrocyte-Neuron Co-Culture Protocol

Primary mixed glial cell cultures were prepared from the brains of postnatal 3–5-day-old (P3–P5) B6 mice, as previously described [12 (link)]. Astrocytes were prepared after removal of CD11b⁺ cells using Dynabeads (Dynal) conjugated with anti-CD11b Ab. The purity of astrocytes was >96% as determined by anti-GFAP and anti-CD11b immunofluorescence. Astrocytes were then seeded into culture flasks, 96-well plates, or Lab-Tek II 8-well chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) at a density of 7.5 × 10⁶ cells per flask, 2.5 × 10⁴ cells per 96-well, or 1.0 × 10⁵ cells per chamber slide well and cultured in the medium described above for 5 days to form a confluent monolayer. LN⁻ cells were seeded on astrocytes at a density of 1.5 × 10⁶ cells per flask or 0.5 × 10⁴ cells per 96-well and chamber slide well and cultured for 7 days.