Primary mouse glial cell cultures were prepared from the cortices of P2-5 pups of C57BL/6 mice as described previously (5557). After careful removal of meninges, single-cell suspensions were obtained by serial trituration of cerebral cortices with 20-gauge (G) and 26G needles with a 10-ml syringe. Cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 15% fetal bovine serum (FBS) and penicillin/streptomycin (all from Thermo Fisher Scientific) and seeded onto T-75 flasks (Corning) precoated with poly-d-lysine (PDL; 25 μg/ml) at about two brains per flask. Medium was changed on day 3 and every 2 to 3 days thereafter. If necessary, then lentiviral infection was performed with glial cells on day 10 as described below. To enrich astrocytes, oligodendrocyte lineage cells and microglia on the surface of mixed glial cell culture were vigorously shaken off, and astrocytes were then collected as negative fractions after magnetic-activated cell sorting (MACS) with CD11b MicroBeads (Miltenyi Biotec). Collected astrocytes were >98% GFAP+ CD11b cells (fig. S6). BV2 microglia were maintained in DMEM/F12 supplemented with 15% FBS and penicillin/streptomycin (all from Thermo Fisher Scientific). Astrocyte-microglia cocultures were prepared by seeding 5 × 105 astrocytes onto PDL-coated six-well plates and adding 5 × 104 BV2 cells 2 days later. Purified astrocyte cultures were prepared by seeding 5 × 105 astrocytes immediately after MACS, onto PDL-coated (10 μg/ml) six-well plates.

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