Microglia Isolation and Polarization Assay

Microglia were cultured from 4 to 6 week old Socs3^fl/fl and DKO mice using a previously described protocol [43 (link)]. Briefly, smashed brain tissue suspensions were filtered through a 70 μm cell strainer (BD Falcon, BD Biosciences). Then the cell suspensions were centrifuged at 600 g for 8 min. The cell pellet was re-suspended in media containing 10% fetal calf serum (FCS), 20 ng/ml M-CSF (Bio-techne, Minneapolis, Minnesota, US), 1% penicillin/streptomycin, 1 mM glutamine in DMEM/F12 (all from Thermo Fisher Scientific, Waltham, MA, USA), and seeded in a 6-well plate (Thermo Fisher Scientific). Floating cells were removed 3 days later and media were changed every 3 days until cells reached 90% confluence. The phenotype of the cells was confirmed by their CD11b and IBA-1 expression (> 90%). The microglia were then treated with 1) M1, or pro-inflammatory stimuli by adding 100 ng/ml IFN-γ (Bio-Techne) and 50 ng/ml LPS (Sigma-Aldrich) or 2) M2, or anti-inflammatory stimuli by adding 20 ng/ml IL-4 (Bio-Techne) overnight. The cells were collected for real-time RT-PCR for cytokine/chemokine gene expression and the supernatants were collected for Luminex assay.