For the tube formation assay, 2 × 104 cells seeded on matrigel matrix-coated (Gibco, USA) wells were treated with 50 ng/ml rhEpiregulin or 200 μg/ml exosomes. After 12 hours incubation, CFSE dye (Dojindo Laboratories, Japan) was added and the optical images of the wells were acquired. Tube-like structures in each well were counted, and results represented the average number of tube-like structures in triplicate wells.
For the vascular permeability assay, endothelial cells were cultured on transwell inserts (0.4 μm pore membrane, Millipore) and allowed to form confluent monolayers (typically after 24 hours). Then complete medium containing 0.12 mg/ml FITC-labeled dextran (40 kDa) (Sigma-Aldrich) replaced the medium in the upper chamber. At each time point after rhEpiregulin or exosomes were added, 50 μl of medium in the bottom chamber was harvested, and fluorescence was measured at excitation 490 nm and emission 520 nm.
For the vascular permeability assay, endothelial cells were cultured on transwell inserts (0.4 μm pore membrane, Millipore) and allowed to form confluent monolayers (typically after 24 hours). Then complete medium containing 0.12 mg/ml FITC-labeled dextran (40 kDa) (Sigma-Aldrich) replaced the medium in the upper chamber. At each time point after rhEpiregulin or exosomes were added, 50 μl of medium in the bottom chamber was harvested, and fluorescence was measured at excitation 490 nm and emission 520 nm.