Transwell Migration and Invasion Assays

Transwell migration and invasion assays were performed using transwell with polyethylene terephthalate membranes (24-well inserts, 8.0 μm, Corning)^{15 (link)}. For migration assay, 200 μL cell suspensions contained 5 × 10⁴ cells was loaded into the upper chamber of a transwell. Next, 600 μL DMEM medium with 10% FBS was placed into the bottom of the well as a source of chemo-attractants. 24 h later, the cells on the lower surface of the insert were fixed with methanol and staining with 0.5% crystal violet. For transwell invasion assay, a similar procedure was performed as the above description except that 1 × 10⁵ cells was loaded into upper chamber pre-coated with matrigel (BD Biosciences, CA, USA). Staining cells were visualized and photographed using a CKX41 microscope (Olympus, Japan). Randomly selected five fields and counted the cells, experiments were repeated three times and the data are presented as the means ± SD.