Microglia Isolation from Young and Aged Mice

Primary microglia were isolated from the brains of young (3–4 months old) and aged (18–20 months old) female C57BL/6N mice as previously described^{16 (link)}. Briefly, we transcardially perfused on mice with sterile 1x PBS. Whole brain tissue was explanted and meninges were removed. Brain tissue was mechanically and enzymatically homogenized into a single cell suspension using the Miltenyi Adult Brain Isolation Kit (130–107-677) per the manufacturer’s instructions. Cell suspensions were strained with a 70 mm cell strainer and then resuspended in 6 mL of 75% stock isotonic percoll (SIP). A density gradient was then made by layering 5 mL of 35% SIP on top of the cell solution followed by 3 mL of 1x PBS. Density gradients were placed on ice for 15 minutes and then spun at 800 × g, 4°C, for 45 minutes with slow acceleration and no break. The top 2 layers were aspirated, and microglia were collected at the 75:35 SIP interface. Cells were then washed in 45 mL of sterile 1x PBS and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (11965–092 Sigma Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Immediately following isolation, primary microglia were seeded in cell culture-treated chamber slides (154941, Thermo Fisher) for immunocytochemistry or 48 well cell culture plates (130187, Thermo Fisher) for flow cytometry at a density of 3×10⁵ cells/cm².