Isolation of Mouse Primary Microglia

Mouse primary microglial cells were isolated from C57BL/6N newborn mice pup brains as described previously (Liao et al., 2016; Chivero et al., 2017). Briefly, brain cortices were dissociated and digested in Hank’s buffered salt solution (14025076, Invitrogen, Carlsbad, CA, United States) supplemented with 0.25% trypsin (25300-054, Invitrogen, Carlsbad, CA, United States). Mixed glial cultures were prepared by resuspending cells in culture medium (DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) with 100 U/ml penicillin, and 0.1 mg/ml streptomycin) as previously described (Guo et al., 2015). Cells were transferred to T-75 cell culture flasks (10 × 10⁶ cells/flask) and incubated at 37°C and 5% CO₂ and half of the cell medium was replaced every 3–5 days. At the first medium change, macrophage colony-stimulating factor (GF026, Millipore, St. Louis, MO, United States) was added (0.25 ng/ml) to promote microglial proliferation. Confluent mixed glial cultures (7–10 days) were shaken at 37°C, 220 × g for 2 h to promote microglia detachment. Cell medium containing released microglia cells was aspirated, centrifuged at 1000 × g for 5 min and collected microglia subsequently plated on cell culture plates for all ensuing experiments. The purity of isolated microglia was confirmed by immunohistochemical staining for Iba1 and was routinely found to be >95% pure (Guo et al., 2015; Chivero et al., 2017).