Assessing Endothelial Permeability with EV Treatment

HUAEC monolayers were cultured on transwell culture insert (1 μm pore, FALCON, #353104) and treated with EVs (Control-EVs or TGF-β-EVs) for 72 h. Permeability assay was performed previously described [25 (link)–27 (link)]. Briefly, the 10 μl of 100 μg/ml FITC-dextran (70 kDa) (Merck, #46945) in the EGM-2MV medium were added into the upper well containing HUAEC monolayer followed by incubation for 30 min. To quantify the HUAEC monolayer permeability, the fluorescence intensity of FITC-dextran leaking into the lower well was measured with FLUOstar OPTIMA-6 (BMG Labtech). The obtained values were averaged and were considered as representative of leakage in HUAEC monolayers related to increased permeability to 70-kDa molecules.