Diic12 3 fluorescent dye

HUVECs were pretreated with VEGF₁₆₅ (25 ng/mL, Sigma–Aldrich) in the presence or absence of anti-VEGF IgG (G6-31) [45] (link) in starvation medium (ECM containing 1% FBS) at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. The tube formation assay was performed using growth factor-reduced Matrigel (BD Biosciences) added to 15-well microslides (Ibidi, Germany); the gel was allowed to solidify at 37 °C for 1 h. Subsequently, subconfluent HUVECs were prestained with 10 μg/mL DiIC₁₂(3) fluorescent dye (BD Biosciences) at 37 °C for 1 h and then harvested with trypsin–EDTA. To evaluate the effects of mouse or humanized QBEND/10, HUVECs were resuspended in starvation medium in the presence or absence of QBEND/10 antibodies at various concentrations and then seeded onto the Matrigel layer at a cell density of 8×10E3 cells per well. After 18 h of incubation, tubular network structures were visualized and photographed using an inverted fluorescence microscope. Cell-covered area and junctions were quantified using ImageJ software.