4.10. Three-Dimensional (3D) Spheroid Sprouting Assay

LECs (3000 cells) were cultured in round-bottom 96-well plates (Nunc, Rochester, NY, USA) precoated with 0.8% agarose for 1 day and the spheroids were collected and embedded in 20% Matrigel-containing (BD Biosciences) fibrin gels (3 mg/mL fibrinogen, 2 U/mL thrombin, and 200 µg/mL aprotinin). The spheroids were cultured for 48 h, fixed with 4% paraformaldehyde for 1 h at room temperature, and stained with rabbit anti-human lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) polyclonal antibody (Abcam, Inc., Cambridge, MA, USA).