Cell Migration and Invasion Assays

Cell migration and invasion assays were performed according to a previous study [18 (link)]. Corning Transwell polycarbonate membrane cell culture inserts with 8.0-μm pores were uncoated for migration assay, or coated with 50 μL of BD Matrigel Basement Membrane Matrix (BD Biosciences, USA) diluted 1:3 with FBS-free DMEM (fetal bovine serum free Dulbecco’s Modified Eagle Medium) for invasion assay. Cells were trypsinized and then resuspended in serum-free medium. 2 × 10⁴ cells were seeded in the top chamber with serum-free medium and allowed to migrate toward serum-containing medium in the lower chamber for 24 h. Then cells were fixed in 4% paraformaldehyde and stained with 0.01% crystal violet (AS1086, ASPEN, China) for 10 min. After wiping the cells remaining in the upper chamber with cotton swabs, the migratory or invasive cells were imaged and counted using a Leica AM6000 microscope (Leica Microsystems, Germany). The numbers of migratory or invasive cells were calculated as the average numbers of five random fields per well under 100× magnification.