2.2.8. Permeability Assay

FITC-labelled dextran was used as the representative of hydrophilic molecules to measure the permeability of endothelial cell monolayer [26,27]. HUVEC were plated in the upper part of a Transwell filter with 0.4 µm pore size at a density of 8 × 1000 cells per well until the formation of a tight monolayer was checked with the microscope. Cells were treated with TNF-α (0.1 µg/mL), and FITC-dextran was added to the top chamber of the Transwell in a final concentration of 1 mg/mL. The culture medium in the upper and in the lower chamber was collected 24 h post-treatment, and fluorescence was measured by fluorimeter (Tecan, Thermo Scientific, Waltham, MA, USA) with an excitation wavelength of 490 nm and an emission wavelength of 520 nm. To evaluate the FITC-dextran passage through the cells monolayer, we calculated the percentage of lower over total fluorescence.
