FITC-dextran paracellular permeability assay

The paracellular permeability of the hCMEC/D3 endothelial barriers was assessed using the FITC-dextran paracellular permeability assay 72 h after transfection as previously described [33, 34]. hCMEC/D3 cells were grown to confluence on transwell polyester membrane inserts (Costar, pore size 0.4 μm; growth area 1.12 cm²) pre-coated with collagen I and fibronectin. After 24h, cells were transfected then left for 72 additional hours, and the apical to basolateral paracellular permeability of the hCMEC/D3 monolayers to (4 or 70 kDa) FITC-dextran was investigated. Briefly, 2 mg/ml of (4 or 70 kDa) FITC-dextran were added to the apical compartment in DMEM media without phenol red containing 2% FBS and incubated for one hour at 37°C. After one hour, 100 μl was collected from the apical and the basolateral compartments and the fluorescence of dextran that passed through the endothelial monolayer to the basolateral chamber measured using the BioTek Synergy H1 plate reader at wavelength excitation/emission: 485/528 nm. The volume cleared was plotted against time, and the slopes of the curves used to calculate the permeability coefficients (Pe, cm/s) of the endothelial cell monolayer as previously described [33]: Pe = PS/s where PS (clearance) is the permeability surface area of the endothelial monolayer and s is the surface area of the filter (1.12 cm²). PS is calculated as follows: 1/PS = 1/me − 1/mf, where me and mf are the slopes of the curves obtained with the endothelial monolayer on filters and with filters only, respectively. me and mf were calculated by plotting the cleared volume against time. The cleared volume is calculated as follows: (AUa−AUb)/ Fi, where AUa is the total fluorescence (arbitrary units) in the basal compartment, AUb is the background fluorescence and Fi is the fluorescence of the initial solution (AU/ml). PS (clearance) is the permeability surface area of the endothelial monolayer and s is the surface area of the filter (1.12 cm²) [33].