Isolation and Characterization of Primary Microglia

Primary microglial cells were isolated from the brain via magnetic cell sorting after conjugation with anti-CD11b antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described [29 (link)]. Isolated CD11b-positive cells (>90% pure as evaluated by flow cytometry) were stained with anti-CD86-FITC (eBioscience, CA, USA), anti-CD68-APC (BioLegend, CA, USA), anti-TSPO-PE (PBR, Abcam, Cambridge, UK), and anti-CD11b-APC-Cy7 (BD Biosciences, CA, USA) antibodies after treatment with the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences). To measure intracellular cytokines, microglia were plated in poly-d-lysine (PDL)-coated 96-well plates (BD Biosciences) and cultured in DMEM/F-12 (Gibco, CA, USA) medium supplemented with 10% fetal calf serum (Gibco) and 100 U/mL penicillium/streptomycin (Sigma-Aldrich, MO, USA) containing a leukocyte activation-cocktail with BD GolgiPlug^TM (BD Biosciences) for 12 h. Microglia cells were treated with the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) and then stained with anti-IL-1β-FITC (eBiosciences), anti-MIP-1α-PE (eBiosciences), anti-TNF-α-APC (BD Pharmingen, CA, USA), and anti-CD11b-APC-Cy7 (BD Biosciences) antibodies. Stained cells were analyzed using a flow cytometer (FACSCanto II; BD Biosciences).