U bottom shaped 96 well plate

Endothelial angiogenesis was studied by spheroid sprouting assay in vitro. HUVECs were transfected with siRNAs or LNA GapmeRs for 24 h. Cells were trypsinized and resuspended in a mixture of culture medium and 0.6 gr/L methylcellulose (Sigma) in a ratio of 80%:20%. Cells were seeded (400 cells per 100 µl) in a U-bottom-shaped 96-well plate (Greiner) to allow the formation of spheroids for 24 h at 37°C. The spheroids were collected, added to methylcellulose (2,4 gr/L) with FBS in a ratio of 80%:20% (Gibco) and embedded in a collagen type I (Corning) gel containing 3,77 g/L collagen I (Corning, USA), 10 % M199 medium (Sigma Aldrich), 0,018 M HEPES (Invitrogen) and 0.2 M NaOH to adjust pH to 7,4. The mixture with the spheroids was allowed to polymerize for 30 minutes in a 24 well plate. Following incubation for 24 h at 37°C with or without VEGFA (50 ng/ml; Peprotech) the gels were fixed with 10% formaldehyde (Roth) and microscope images were taken at 10x magnification (AxioVert microscope, Zeiss). The cumulative length of sprouts was quantified using the image analysis software ImageJ.