Transwell invasion and migration assay

For invasion assay, thaw Matrigel (Corning Inc., Kaiserslautern, Germany) on ice in 4 °C refrigerator overnight. Dilute Matrigel in serum free DMEM to final concentration of 0.5 mg/ml. Load 70 μl of Matrigel into the Transwell chamber with 8.0-µm pore size polycarbonate membranes (Corning Inc, Corning, NY, USA) and solidified in a 37 °C incubator. After 24 h, add 200 μl cell suspension (5 × 10⁴ cells/ml in serum freem DMEM) onto Matrigel-coated cell culture insert and add 550 μl DMEM complete media containing 10% FBS into lower chamber. After 24 h, remove the media and Matrigel in upper chamber and wash twice by PBS. Fix cells with 4% formaldehyde for 1 min at room temperature. Remove formaldehyde and wash twice with PBS. And then the invaded cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for visualization of the nuclei. The invasive cells were detected by fluorescence microscope at × 100 magnification. The stained nuclei were counted and quantified using ImageJ software 1.45 s (U.S. National Institutes of Health, Bethesda, MD, USA).

For migration assay, load 200 μl cell suspension (5 × 10⁴ cells/ml in serum freem DMEM) onto Transwell chamber with 8.0-µm pore size polycarbonate membranes and add 550 μl DMEM complete media containing 10% FBS into lower chamber. After 18 h, remove the media and wash twice by PBS. Fix cells with 4% formaldehyde for 1 min at room temperature. Remove formaldehyde and wash twice with PBS. And then the invaded cells were stained with DAPI for visualization of the nuclei. The migrated cells were detected by fluorescence microscope at × 100 magnification. The stained nuclei were counted and quantified using ImageJ software 1.45 s (U.S. National Institutes of Health, Bethesda, MD, USA).