Microglia Isolation and Characterization

From the same harvest, macrophage precursors (pMacpre) were either lysed directly or differentiated to microglia in monoculture (pMGL) or microglia in co-culture with MNs (co-pMG) for 14 days. pMGL were rinsed with PBS and directly lysed in the dish. For both pMacpre and pMGL, RNA was extracted using an RNAeasy Mini Plus kit (Qiagen) according to the manufacturer’s instructions. Co-cultures were first dissociated by 15 min incubation with papain (P4762, Sigma-Aldrich) diluted in accutase (20 U/mL) and gentle trituration based on a previously published protocol^{40 (link)}. The cell suspension was then passed through a cell strainer (70 μm, Falcon) to remove cell clumps. To extract co-pMG, magnetic-activated cell sorting (MACS) was then performed using CD11b-MACS beads (130–093-634, Miltenyi Biotec) according to the manufacturer’s instructions. The panned cell population was lysed for RNA extraction using an RNAeasy Micro kit (Qiagen) according to the manufacturer’s instructions. In addition, RNA from human fetal microglia and blood monocytes from three different healthy genetic backgrounds was re-used from our previous study^{26 (link)}.