ECs were grown to confluency on Transwell inserts (EMD Millipore, Kankakee, IL) containing 3 μm pores. Then, 10 μg/ml of FITC-labeled dextran (3 kD) was added to the media over the cells along with either vehicle or 40 μM of hemin. After 30 min, 100 μl of media was extracted from below the Transwell insert for measurement of FITC concentration by fluorometry (with excitation and emission spectrum peak wavelengths of 495 nm and 519 nm, respectively) to determine the degree of EC monolayer permeability in either condition, as described previously (23).
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