Matrigel precoated 96 well plates

Firstly, 96-well plates used for microvascular (capillary) network and sprouting formation assay were precoated with Matrigel (BD, Biosciences) as previously described by Li (Li et al., 2015 (link); Li et al., 2021 (link)). The cells were pretreated with growth factors, inhibitors, or siRNA-mediated knockdown for 24 h; and transferred onto Matrigel-precoated 96-well plates (BD, Biosciences) (1 × 10⁵/well); and then cultured at 37°C in 5% CO₂ for further 5 and 24 h (for microvascular network and sprouting formation assay, respectively). Images were taken under an inverted microscope. The capillary and sprout lengths were measured using an angiogenesis analyzer (ImageJ) as previously described (Li et al., 2021 (link)).

Tube formation assays were performed to evaluate the angiogenic ability of endothelial cells. Briefly, RF/6A cells (1 × 10⁴ cells/well) were seeded onto Matrigel-precoated 96-well plates (BD Biosciences, USA). Then, these cells were cultured at 37 °C for 8 h. Finally, the tube formation was observed using an inverted microscope (Nikon, Tokyo, Japan).