Quantifying HUVEC Migration and Adhesion

HUVEC migration was assessed, using CIM-plates (Roche) and the RTCA system (ACEA Bioscience); adhesion was assessed using E-plate (Roche) in the RTCA system^{55 (link)}. Briefly, the membrane of the top chamber of a CIM-plate was coated with fibronectin by adding 40 µL of 20 µg mL⁻¹ fibronectin dissolved in PBS and incubating at 37°C for 30 min. 180 µL of EGM-2 (complete media for HUVECs) or EBM (serum free media) or MB231-LEC CM was added to the bottom chambers. The equilibrated plate was removed from the incubator and 100 µL of the trypsinized cells (45,000 HUVECs per well) with or without inhibitors were added to the top chamber. After 30 min incubation at room temperature, the stabilized chamber was loaded in the RTCA machine and the cell index was measured continuously for 20 h. Cell indices at 20 h were selected for analysis. ACEA E-plates (Roche Diagnostics) were used to measure the extent of HUVEC adhesion. Briefly, HUVECs (25,000 cells per well) in 100 µL of EGM-2 (complete media for HUVECs) or EBM (serum free media) or MB231-LEC CM were added. After equilibrating at room temperature for 30 min, the E-plate was loaded into the RTCA personal system. Cell indices at 3 h were analyzed.