hLEC tube formation assay

Tube formation assay was performed to examine the effects of rhVEGF-C on the tube formation of hLECs in vitro. Ten μL/well liquid Matrigel (Corning, Cat. #356243, Bedford, MA, USA) was coated on the inner walls of 15-well Ibidi μ-slides for angiogenesis (Ibidi, Martinsried, Germany) (Gagliostro et al., 2016), and polymerized for 1 hour at 37°C. hLECs were synchronized by serum starvation for 12 hours prior to treatment with equal amounts of purified factor (100 ng/mL) or 10% fetal bovine serum for 48 hours (Joukov et al., 1998; Chien et al., 2009). hLECs (1 × 10⁴/well) in 50 μL media were seeded into the upper well and incubated for 12 hours at 37°C to allow tube formation. Images of tube formation of each chamber were captured using an inverted phase contrast microscope (Leica DMI 4000B, Wetzlar, Germany) at 5× magnification. Total lengths of tube-like structures (exceeding 20 mm in diameter) were measured and quantified using ImageJ (NIH, Bethesda, MD, USA) (Kazenwadel et al., 2012; Shin et al., 2015).