Isolation of Adult Microglia from Mice

Adult microglia were isolated from 3-4-month-old wild-type (for protein isolation) or Cx3cr1^GFP/+ mice (for phagocytosis assay) using a protocol adapted from Bohlen et al.^{64 (link)} Mice were euthanized by CO² asphyxiation, and brain regions were immediately removed. Tissue was enzymatically and mechanically dissociated using the Miltenyi neural dissociation kit (130-092-628, Miltenyi, Auburn, CA) following manufacturer’s recommendations. Myelin removal was performed using myelin removal beads II (130-096-733, Miltenyi) with LS columns (130-042-401, Miltenyi) following manufacturer’s recommendations. After myelin removal, the microglia were selected by anti-CD11b-coated microbeads (130-093-636, Miltenyi) with the QuadroMACs separator following manufacturer’s recommendations. Cells were manually counted with a hemocytometer using trypan exclusion staining and were either plated at 56,000/cm² in a 48-well plate with DMEM supplemented with 10% FBS (Sigma F4135) and 1% Penicillin-streptomycin (Gibco 15140) for 5 hours for phagocytosis assay or pelleted and immediately frozen on dry ice for protein isolation.