In vitro angiogenesis kit

The In vitro Angiogenesis Kit (Millipore) was employed to evaluate endothelial angiogenic properties. HUVECs were transfected and seeded at a density of 2 × 10⁵ cells/well in a 6-well plate and allowed to grow to ∼75% confluency. The kit-provided matrix solution was added into designated wells of a 96-well plate. Transfected cells from the previous preparation were then harvested and seeded at an equal density of 1 to 1.5 × 10⁴ cells/well onto the designated wells in EGM-2 supplemented with LPS or vehicle diluent. Phase contrast microscopy was employed (Optika) to obtain pictures of cells under phase contrast in each designated well over time to monitor tube formation, and quantification was performed according to the manufacturer’s instruction.

ECMatrix (ECM625, Merck KGaA, 64293 Darmstadt, Germany) was thawed at 4 °C overnight. Then, pre-chilled 96-well plates were incubated with 50 µL diluted ECMatrix at 37 °C for 1 h for ECMatrix to solidify. Next, 150 µL of human vascular endothelial cells (HUVECs) at 1 × 10⁴ with or without UA were added to the solidified matrix and incubated at 37 °C for 12 h. Afterward, endothelial cell formation was assayed with the in vitro angiogenesis kit (Millipore, Billerica, MA, USA) using a microscope. The focus was placed on distinct areas, and the tubes that formed were counted according to the kit’s instructions.

The in vitro angiogenesis kit was ordered from Millipore (Billerica, MA, USA). The EC Matrix was thawed at 4 °C overnight. The required wells of the pre-chilled 96-well plates were coated with diluted EC Matrix (50 μL) and incubated at 37 °C for 1 h to solidify. Human umbilical vein endothelial cells (HUVECs; 1 × 10⁴; 150 μL) with or without silibinin were added to the solidified matrix and incubated at 37 °C for 8 h. Endothelial cell formation was observed using a microscope. The focus was placed on distinct areas, and the tubes formed were counted according to the kit procedure.

The In vitro Angiogenesis Kit (Millipore) was employed to evaluate endothelial angiogenic properties. HUVECs were transfected with sieNOS and seeded at a density of 2 x 10⁵ cells/well in a 6-well plate and allowed to grow to ~75% confluency. The kit-provided matrix solution was added into designated wells of a 96-well plate. Transfected cells from the previous preparation were then harvested and seeded at a density of 1–1.5 x 10⁴ cells/well onto the designated wells in EGM-2 medium. Phase-contrast microscopy was employed (Optika) to take pictures of cells under phase-contrast in each designated well over time to monitor formation of tube-like structures in vitro. Mesh area and tube-length was measured using angiogenesis analyzer software from Image J.

MSCs were cultured in endothelial differentiation medium for 7 d and capillary tube formation was then induced using an in vitro angiogenesis kit (Chemicon). Briefly, basement membrane-like material (EC Matrix; BD) was diluted to 0.5–0.7 mg/mL in endothelial differentiation medium. A total of 5 × 10⁴ cells were seeded in 300 μL of 0.5–0.7 mg/mL Matrigel in each well of a 24-well plate. The Matrigel-cell suspension was polymerized for 4 h at 37°C. Then, 300 μL of endothelial differentiation medium supplemented with 50 ng/mL VEGF was added, and the gel-embedded cells were cultured at 37°C and 4% CO₂. The structures were photographed using a phase contrast microscope (Olympus). Total cord length was quantified using image-Pro Plus v4.5 software.