Inflammatory Markers of BBB Endothelial Cells

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.