Matrigel matrix 356237

Matrigel Matrix 356237 (Corning Inc., Life Sciences, MA) was coated on 15-well μ-angiogenesis slides at 10 μL/well (ibidi GmbH, Planegg, Germany). The coated slides were incubated for 15 minutes at 37°C. Treated HUVECs (10,000 cells/well) were harvested and seeded into the Matrigel-containing wells and incubated for 6 hours at 37°C to allow tube formation. The wells were then imaged for capillary-like structures using an inverted microscope (Life Technologies). Quantification of the tubes was performed by taking 4 images of each chamber, which were then analyzed by ImageJ for in vitro angiogenesis [33 (link)].

In vitro tube formation was performed with Matrigel Matrix 356237 (Corning Inc., Life Sciences, MA) as per manufacturer’s recommendation. 96-well plates were precoated with Matrigel (50 μl/well), which was allowed to solidify at 37°C for 60 min. HUVECs were exposed to the vehicle or FMNT (10, 20 and 40 μM) for 24 h or pretreated with L-NAME (100 μM), or LY294002 (20 μM) or PD98059 (10 μM) for 1 h before incubation with FMNT (20 μM) for 24 h. Cells cultured with VEGF (40 ng/ml) were used as a positive control. After the treatment, cells were harvested, resuspended and plated on Matrigel (20,000 cells/well) at 37°C for additional 3 h or 5 h. Quantification of the tubes was performed by analyzing three images of each well with a microscope (Moticam 5+) at 100× magnification, and the closed networks of vessel-like tubes were counted in each image with the average to be used for data analysis. The experiment was repeated at least three times to determine tube formation for each treatment group. The tube formation was expressed as the percentage over the vehicle control.