Efluor 520

Brains were dissected into 1-2 mm 3 pieces with small scissors and incubated for 30 min at 37°C in 1 ml HBSS solution containing 2% FBS, 1 mg/ml Collagenase D and 50 µg/ml DNase I. 10 mM EDTA was added to the 1.5 mL tube to stop the enzymatic reaction and homogenates were pipetted for further dissociation. Next the homogenates were ltered through a nylon mesh (70 µm pore size) in a 50 mL tube using the plunger of a 10 mL syringe, washed with cold FACS buffer (2% FBS, 1 mM EDTA in PBS without Ca 2+ or Mg 2+ ) and centrifuged at 300g at room temperature for 5 min. For the enrichment of microglia, the cell pellet was resuspended with 30%-37%-70% Percoll density gradient medium and centrifuged at 800g, no acceleration and braking, at room temperature for 40 min. Next, the 70-37% density gradient interphase containing the macrophage subpopulations cells was collected into a clean tube, washed with 5 ml FACS buffer and centrifuged at 500g at 4°C for 5 min, followed by antibody labeling and ow cytometry analysis. Antibodies against PerCP-CD11b (Biolegend, San Diego, CA), PE-CD45 (Biolegend, San Diego, CA), CD16/32 (Biolegend, San Diego, CA), xable viability dye eFluor 520 (ebioscience, San Diego, CA) were used. Analysis was performed on a Calibur (BD Biosciences, BD Diva Software) and analyzed with FlowJo software.