Transwell invasion assay

Transwell invasion assay was performed using 24-well Transwell plates with 8-µm pores and a coating of Matrigel (100 µl/well) (BD Biosciences, San Jose, CA, USA) to measure the invasion status of A549 cells. In addition, 2×10⁴ cells were placed in the upper Transwell chamber. Then, 600 µl complete DMEM was added in the lower chamber as the chemo-attractant. After cells were incubated for 24 h at 37°C, non-invasive cells in the top chamber were removed with cotton swabs. Invasive cells at the bottom of the membrane were fixed with methanol and then were stained with crystal violet. The number of invasive cells was counted by hemocytometry to assess the invasion status of the cells.