4.11. Invasion Assay

We performed an invasion assay using Transwell Corning® BioCoat™ Matrigel® Invasion Chambers with 8.0-μm polyester (PET) membrane in 6-well Plates (Corning Costar Corp., Cambridge, MA, USA). Cells (1 × 10⁵) were plated onto Transwell chambers with serum-containing media and incubated at 37 °C in 5% CO₂ for 24 h. After 24 h, we removed the cells from the top of the Transwell chambers using a cotton swab (residual cells). We stained the membrane using a Hema 3 kit (Fisher Scientific Company LLC, Kalamazoo, MI, USA), as per the manufacturer’s instructions. Cells were counted in five independent high-powered fields (200×) using a Nikon microscope.