Matrigel invasion assay

Transwell chambers (Costar, Cambridge, MA, USA) with a pore size of 8 μm were used to detect the invasive ability of the cells. Matrigel (100 µL of a 1:7 dilution, BD Biosciences, CA, USA) was added to each insert, and the plates were placed in a 37°C incubator for at least 2 h. Twenty-four hours after transfection, 8 × 10⁴ cells were added to 100 μL medium supplemented with 2% FBS and seeded into the upper chamber. Medium supplemented with 20% FBS as the chemoattractant was added into the lower chamber. After incubation for 20 h, the non-invading cells on the top surface of the filters were swabbed with a cotton swab, and the cells adhering to the lower surface of the filters were fixed with 4% paraformaldehyde and stained with hematoxylin. Five high-power fields were selected randomly under a microscope to count the number of invasive cells.