Transwell invasion assay

Cell invasion was performed using 24-well transwell plates (Corning) pre-coated with Matrigel (BD). Cells were seeded in the upper chambers of inserts at a density of 5 × 10⁴ cells per well with serum-free DMEM. DMEM supplement with 10% serum was added to lower chambers. After being cultured for 48 h, invasion cells were stained by violet crystalline solution according to the protocol. In order to count cell numbers in five random fields, phase contrast microscopy was employed.