8.0 mm pore polycarbonate membranes

As a classical in vitro model of angiogenesis, migration was assessed in 24-well plates with 8.0-mm pore polycarbonate membranes inserted (Corning, United States). In brief, we added 800 μl of EGM-2 into each well of the 24-well plate, which was followed by inserting chambers with polycarbonate membranes. Then, 2 × 10⁴ cells with 200 μl of EBM-2 were added into each chamber in triplicate. After further incubation for 20 h, cells were fixed with PFA and stained with crystal violet for 10 min. The images of cells that migrated across the membrane were captured using a microscope (Leica, Germany). The tinctorial cells under the membranes were then counted and analyzed.

Migration assays were performed in a 24‐well Transwell chamber with 8.0‐mm pore polycarbonate membranes (Corning, NY, USA). For cell invasion assay, the same membranes in the upper chamber were precoated with 100 μL of 1 mg·mL⁻¹ Matrigel solution (BD Biosciences, San Jose, CA, USA) at 37 °C for 30 min in an incubator. For both migration and invasion assays, 5 × 10⁴ cells in serum‐free medium were placed in the upper Transwell chamber. After 24 h, the cells on the upper surface of the membrane were removed, and the cells on the lower surface were fixed and stained with 0.1% crystal violet. The cells in three random microscopic fields were counted and imaged in a light microscope with a DP70 CCD system (Olympus Corporation, Shanghai, China).