Transwell plates with 8 m pore filters

Cell invasion assay was performed using 24-well Transwell plates with 8-µm pore filters (Corning Life Sciences; cat. no. 3422) precoated with Matrigel Basement Membrane Matrix (BD Biosciences) at 37°C for 1–4 h according to the manufacturer's protocols. Briefly, 1×10⁵ cells were suspended in 100 µl of serum-free medium and added to the upper chamber and then 600 µl RPMI-1640 supplemented with 20% FBS was loaded into the lower chamber. After 24 h of incubation, the non-invaded cells in the top chamber were removed with a cotton swab and the cells fixed with 100% methanol for 20 min at room temperature and stained with 0.1% crystal violet (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. G1063) for 30 min at room temperature. The stained cells were imaged under an inverted light microscope (DMi8; Leica Microsystems GmbH; magnification, ×100) and analyzed using ImageJ software.

Cell migration assays were performed using Transwell plates with 8-µm pore filters (Corning, Inc.), following the manufacturer's protocol. Briefly, for the cell migration assay, cells (2×10⁵) were suspended in 200 µl of serum-free medium and seeded into the upper chambers of the Transwell plates; serum-free medium supplemented with 10 ng/ml VEGF-A was applied to the lower chamber as a chemoattractant to induce migration. After incubation for 24 h at 37°C, cells were fixed with 4% cold paraformaldehyde for 15 min at room temperature and stained with 0.1% crystal violet solution for 5 min at room temperature. Non-migrating cells remaining on the upper surface of the filter membrane were scraped off gently with a cotton swab. For quantification, the stained cells that had migrated to the other side of the membrane were extracted with 33% acetic acid. The absorbance of the eluted stain was determined at 570 nm.