Trans‐Endothelial Permeability Assay

A transendothelial permeability assay was performed using the Vascular Permeability Assay Kit (Millipore, Billerica, MA, USA) to determine the permeability of a labeled tracer flux across the confluent HUVECs grown on polycarbonate filters. Briefly, HUVECs were seeded onto the collagen‐coated inserts and grown for 72 h to reach confluence and to form an endothelial monolayer in order to occlude the membrane pores on Transwell inserts. After experimental manipulations, the solution of FITC‐labeled Dextran was applied onto the top of the insert for 20 min at room temperature, allowing the fluorescent molecules to pass through the endothelial monolayer at a rate proportional to its permeability. The extent of permeability was determined after 60 min by measuring the fluorescence intensities of FITC in the culture media derived from the receiver tray wells in a microplate reader FL 800 (BioTek, Winooski, VT, USA) equipped with a filter set (excitation at 485 nm; emission at 535 nm). The data are expressed as relative luminescence unit (RLU).