Anti cd11b coated microbeads

Animals were sacrificed by cervical dislocation, and the hippocampi were promptly dissected from the brains of the control mice and CUMS mice, followed by a prechilled PBS wash. Tissue dissociation was performed based on established protocol [22 (link)] with minor modifications. In brief, the tissues were digested with Papain (2 mg/mL) (Worthington, Colorado, USA) in RPMI 1640 medium at 37°C for 30 min. The dispersed cells were then passed through a 70 μm nylon mesh and collected through centrifugation at 300 g for 6 min at 4°C. After removing red blood cells using Red Blood Cell Lysis Buffer (Solarbio, Beijing, China) added with 10 µL DNase I (Sigma, Michigan, USA), total cell pellets were resuspended with PBS followed by microglia isolation. Microglia were isolated using anti-CD11b-coated MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) with a MACS multi-stand separator as per the manufacturer’s instructions. The isolated cells were then subjected to RNA extraction.

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.