8 μm pore 24 well transwell plates

Cell invasion was measured using 8-μm-pore 24-well transwell plates (Corning, New York, US, #3422). The upper layer was inoculated with serum-free cells, and placed in medium with 10% serum. After 12 h of culture, cells were fixed with 90% ethanol, wiped dry, and dyed with 1% crystal violet. Migrated cells were counted by selecting three fields per filter (20× magnification).

The cell migration and invasion capabilities were assessed by transwell assay with 8-μm pore, 24-well transwell plates (Corning). For the migration assays, 10 × 10⁵ Caco-2 LV-DKK4, HCT8 LV-DKK4, HCT116-LV-shDKK4, SW480 LV-shDKK4, or corresponding negative control cells were seeded in the top chamber lined with an uncoated membrane (Millipore). In the invasion experiment, 10 × 10⁵ of the aforementioned cells were plated in the upper chamber coated with Matrigel (BD Biosciences). After incubation at 37 °C with 5% CO₂ for an appropriate duration, cells that had migrated or invaded through the membrane were stained with 0.1% crystal violet and counted under a microscope (Olympus) to determine their relative numbers.