2.4. Isolation of primary microglia

Murine primary microglia were obtained from ICR pups (<3 days old, raised in pathogen-free laboratory environment). Brains were excised and placed in 75% ethanol. The cerebellum was isolated before the brains were transferred into Hank’s Balanced Salt Solution (HBSS; Meilunbio). Cortices and meninges were removed. The remaining brains were washed by phosphate buffer saline (PBS; Meilunbio) and digested in 0.25% Trypsin/EDTA (Thermo Fisher Scientific) and 2.5 μg/mL DNase I (Beijing Dingguo Changsheng Biotechnology) for 5 min followed by gentle trituration. Tissue homogenates were passed through a sieve (75-µm mesh) and collected by horizontal centrifugation for 2 min at 2,000 rpm, then resuspended in 12% FBS-F12DMEM medium (Thermo Fisher Scientific) for plating. On the second day after isolation, primary microglia cultures were centrifuged for 2 min at 2,000 rpm. Half of the supernatant was transferred back to the plate, and an equal volume of fresh 12% FBS-F12DMEM was added. The medium for microglia culture was changed every 3 − 4 days. After 14 days, microglia cells were gently shaken off, and were re-plated in 12% FBS-F12DMEM for 2 − 3 days prior to further immunoblotting, immunofluorescence staining or flow cytometry analysis.