Microglia and other immune cells within the CNS were isolated by a one-step density centrifugation protocol as described previously, with some modifications64. Briefly, mice were euthanized with CO2 inhalation and subsequently perfused with ice-cold PBS by cardiac puncture, and brains were collected and placed in RPMI medium (Thermo Fisher Scientific) on ice. Tissue was homogenized with a tissue dounce grinder set. Homogenates were resuspended 37% PercollTM (Sigma) and subjected to density centrifugation (550×g, 18 min) to remove myelin debris. Cells were collected in 1x HBSS (Thermo Fisher Scientific) and used for flow cytometry. Microglial cells were defined as CD11b+CD45intCX3CR1+, whereas the CD45hi population represented infiltrating lymphoid (CD11b-) and myeloid (CD11b+) cells.
