Primary Neuron and Microglia Co-culture

Primary neurons were derived from the cerebral cortex of p0–p1 mice following standard operational procedure using the neural tissue dissociation kit—postnatal neurons (Cat. 130-094-802, Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany), as previously described [30 (link)]. In brief, the brain cortices from 6 mice of both sexes were pooled as a single experimental group and subjected to enzymatic and mechanical dissociation, then 150,000 primary neuronal cells were seeded for each well of a poly-L-ornithine-coated 24-well plate, replacing half of the medium volume every 2 or 3 days. At day 10, 37,500 primary microglia cells isolated from the whole brain of adult male or female mice (age 3–6 months) were seeded on a neuron layer [19 (link)]; briefly, the brains from two mice were pooled and subjected to enzymatic and mechanical dissociation and microglia were purified using a magnetic column and anti-CD11b-coated microbeads (Cat. 130-093-634, Miltenyi Biotec). Neuronal and microglial cultures were grown in Neurobasal A medium (Cat. 10888-022, LifeTechnologies, Carlsbad, CA, USA) containing 1% streptomycin–penicillin, 1% GlutaMAX, 2% B-27 Supplement (Cat. 17504-044; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 10 mM HEPES (Cat. H0887, Merk, Darmstadt, Hesse, Germany), in a humidified 5% CO₂-95% air atmosphere at 37 °C.