In vitro endothelial cell permeability assay

Transendothelial electrical resistance (TEER) was measured by Electric Cell-substrate Impedance Sensing (ECIS). An 8W10E+ electrode 96-well culture array (Applied Biophysics) was coated with 0.1% gelatin (Sigma) and 10 μg/mL human fibronectin. 1 x 10⁴ primary MBMECs isolated from EphA2+/+ and EphA2-/- mice per well were seeded in complete media onto the electrode culture array and monitored until a stable monolayer formed. Once a stable measurement of resistance from MBMECs was achieved, resistance changes were further monitored in real time for up to 48 hours. Data were analyzed using the X-CELLigence experiment report software.

To assess paracellular permeability, primary MBMECs from EphA2+/+ and EphA2-/- mice were seeded on gelatin-coated transwell filter plates (Costar 3413, 0.4μm pore size; Corning) and grown to confluence. For stimulation, MBMECs were serum starved for 4 hours in iDMEM/F12 containing 1% BSA, then pre-incubated with the lysates of naïve RBCs and PbA-schizonts (pRBCs), 5μg/mL of cluster-activated ephrin-A1-Fc and EphA2-Fc (for 2 hours) parallel to the diffusion of 25μg/mL FITC-dextran (250kD; Sigma-Aldrich) at 37°C and 5%CO₂. Fluorescence in the lower chamber was measured with a plate reader (BioTek) at 490nm.