Dmi8 automated platform microscope

Growth factor-reduced Matrigel (Corning, Corning, NY, USA) was plated into a 96-well μ-angiogenesis plate (ibidi, Planegg, Germany) at 10 μl/well, and incubated at 37°C in 5% CO₂ in air for 60 min as described (14 (link)). HUVECs were detached (above) and resuspended in complete endothelial cell growth medium 2 (EGM-2) (PromoCell), and seeded at 10,000 cells/well in medium supplemented with 10 µM sulforaphane (SFPH, Sigma) (negative control), 1.2 nM VEGF-165 (Novus Biologicals) (positive control), or 5, 10, 20, and 40 nM Ov-GRN-1. The ibidi plate was incubated for 12 h in a humidified atmosphere of 5% CO₂ in air at 37°C in a microscope stage top incubator (OKOLAB, Pozzuoli, Naples, Italy). At intervals, photomicrographs of cells and nascent and developed tubules were collected using a Leica DMi8 automated platform microscope under bright field at 2.5 × magnification, and Leica LASX software (Leica).

Growth factor-reduced Matrigel (Corning, Corning, NY, USA) was plated into a 96-well μ-angiogenesis plate (ibidi, Planegg, Germany) at 10 μl/well, and incubated at 37 °C in 5% CO₂ in air for 60 min as described [18 ]. 3B-11 cells were detached using Trypsin/EDTA and resuspended in DMEM (Gibco), and seeded at 30,000 cells/well in medium supplemented with 10 μM sulforaphane (SFPH, Sigma) (negative control), or 1.8 μg/mL or 3.6 μg/mL IPSE. The ibidi plate was incubated for 5 h in a humidified atmosphere of 5% CO₂ in air at 37 °C in a microscope stage top incubator (OKOLAB, Pozzuoli, Naples, Italy). At intervals, photomicrographs of cells and nascent and developed tubules were collected using a Leica DMi8 automated platform microscope under bright field at 2.5 × magnification, and LASX software (Leica).