Isolation and Culture of Murine Microglia

Adult C57BL/6 mice were perfused with ice-cold Hank’s Balanced Salts Solution (HBSS) and brains kept in cold HBSS. After removal of cerebellum and meningeal layers, brains were diced, and enzymatically processed using as per manufacturer’s instructions using a MACS Neural Tissue Dissociation Kit (P) (Miltenyi Biotech, Bisley, UK). Brains were then mechanically homogenised using a Dounce homogeniser, and myelin removed from the resulting cell suspension using a one-step 30% Percoll gradient^{31 (link)}. Microglia were then isolated using magnetic CD11b + beads (Milteny Biotech), and seeded on poly-L-lysine coated plates (~1.7 × 10⁵ cells/ml), and maintained with DMEM (with 10% fetal bovine serum and 1% penicillin streptomycin) supplemented with 10 ng/ml of recombinant mouse M-CSF (R&D systems, Abingdon UK), and 50 ng/ml of recombinant human TGF-β. Media was changed at 3 days, and cells treated after 7 days in culture.