Endothelial Cell Monolayer Permeability Assay

In vitro endothelial cell monolayer permeability was assessed as previously described [28 (link)]. Briefly, HRECs were grown to confluence on gelatin-coated inserts of Transwell Permeable Supports (0.4 µm, Costar, Corning, NY, USA) and incubated at 37 °C for 6 days in low-glucose, high-glucose, or HGM conditions. The treated cells were then probed with 1 mg/mL 40-kDa FITC-dextran for 60 min, and the amount of FITC-dextran in the lower chambers of the culture wells was measured by a fluorescence scanner (InnoScan 300, Innopsys, Carbonne, France) using well-type arrays (n = 4), fabricated by mounting PDMS gaskets onto clean glass slides as previously described [29 (link)].