Anti il 1β fitc

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).

Surrogate markers of inflammation, oxidative stress and monocyte recruitment of BBB ECs were determined by using flow cytometry as established previously (Elahy et al., 2015 (link)). Briefly, the single cell suspensions were incubated with extracellular markers of anti-CD31-BV421 (1:200), anti-CD45-PerCP-Cy5.5 (1:500), anti-VCAM-1-APC (1:200, Biolegend) and anti-ICAM-1-PE (1:200, Biolegend) for 30 min at 4°C. Following the permeabilization, the cells were incubated with anti-TNF-α-APC (1:50, biolegend), anti-IL-1β-FITC (1:50, eBioscience, CA, USA) and dihydroethidium (DHE, 3.0 μg/L, Sigma-Aldrich) for 30 min at 4°C. Samples were acquired through FACS Canto II with a gating for CD31^posCD45^neg. The expression of each protein of interest was determined by calculating the fluorescent intensity per EC.