Isolation of Adult Mouse Microglia

Adult microglia isolation was performed using MACS, as previously described [47 (link)]. Briefly, mice were anesthetized with avertin and transcardially thoroughly perfused with PBS to remove circulating blood cells in the CNS. Each dissected brain was chilled on ice and then minced in enzymatic digestion buffer containing 0.2% Collagenase Type 3 (Worthington, LS004182) and 3 U/mL Dispase (Worthington, LS02104). Minced brain tissue was then incubated at 37°C for 45 min. The enzymatic digestion was stopped with inactivation buffer containing 2.5 mM EDTA (Thermofisher, 15575020) and 1% fetal bovine serum (Invitrogen, 10082147). The digested brain tissue was then triturated in a serological pipette several times before passing through a 70-μm filter. The homogenate was then depleted of myelin using myelin removal beads (Miltenyi Biotec, 130-096-733) and magnetic LD column (Miltenyi Biotec, 130-042-901). The elute was enriched for microglia with CD11b magnetic beads (Miltenyi Biotec, 130-049-601) and MS column (Miltenyi Biotec, 130-042-201).