Isolation and culture of primary murine microglia

Microglia cells from either newborn (P0-1, male or female) or adult (P40-55, male) C57BL/6J, NKCC1^fl/fl (WT), or NKCC1^{fl/fl ΔCx3CR1} (KO) mice were isolated by magnetic separation using anti-CD11b microbeads (Miltenyi Biotec, Germany), with slight modification of the protocol described by Otxoa-de-Amezaga [85 (link)]. After transcardial perfusion with ice-cold phosphate-buffered saline (PBS), the brain tissues (cortices and hippocampi) were enzymatically dissociated with Neural Tissue Dissociation Kit-P (#130-092-628; Miltenyi Biotec). Myelin was removed by MACS Myelin Removal Beads II (#130-096-733, Miltenyi Biotec), then cells in a single-cell suspension were magnetically labeled with MACS CD11b microbeads (#130-093-634, Miltenyi Biotec) and were separated using MS columns (#130-042-201, Miltenyi Biotec, Germany). Cells selected with CD11b microbeads were plated onto poly-L-lysine precoated 96-well or 386-well plates at 3 × 10⁴ cell/cm² density and were cultured at 37°C in a 95% air/5% CO₂ incubator in DMEM/Glutamax medium (#31966–021, Gibco) supplemented with 10% fetal bovine serum (FBS, #FB-1090, Biosera), 1% Pen/Strep (10,000 U/ml; #15140–122, ThermoFisher Scientific), and 10 nM macrophage colony-stimulating factor (M-CSF; #PMC2044, ThermoFisher Scientific) for 10 days.