Cell Migration and Invasion Assay

A cell migration assay was carried out using the Boyden chamber technique, as previously reported^{35 (link)}. Cell culture inserts (8-μm pore size, 6-mm in diameter) were used according to manufacturer’s instructions. Invasion assays were performed using Corning Matrigel invasion chambers (pore size: 8 μm; Discovery Labware Inc., Woburn, MA, USA) as previously reported^{21 (link)}. Cells were plated at a density of 1 × 10⁵ cells/500 μL on the upper surface of the inserts, and 4 h later for migration and 16 h later for invasion, cells that had migrated through the membrane to the lower surface of the filter were fixed and stained with a Diff-Quik staining kit (Polysciences, Inc., Warrington, PA, USA). The images were taken using a Mantra multi-spectral microscope (Perkin-Elmer), and then the images were loaded into inForm software ver. 2.4 (Perkin-Elmer) to count the number of migrated or invasive cells in 12 random fields under a 20 × magnification objective. All independent experiments were repeated twice in triplicates.