2.7. Transwell and Boyden Assays

In vitro cell migration Transwell assay and invasion Boyden assay were performed according as previously described [14]. For the Transwell assay, 1 × 10⁴ cells in 100 μl DMEM medium without FBS were seeded onto the upper chamber of the Transwell apparatus (Costar, MA), and 500 μl DMEM with 10% FBS was added into the lower chamber as a chemoattractant. After incubation for 6 hours at 37°C in a 5% CO₂ atmosphere, the fibronectin-coated polycarbonate membrane insert was washed with PBS, and cells adhering to the top surface of the insert were removed with a cotton swab. Cells on the lower surface were then fixed with methanol, stained with crystal violet solution, and counted under a microscope in five predetermined fields (200x). All assays were independently repeated at least thrice. For the Boyden assay, the procedure was similar to the above one, except for the fact that the Transwell membranes were first precoated with 24 μg/μl Matrigel (R&D Systems, USA), and the cells were incubated in the Transwell apparatus for 8 hours at 37°C in a 5% CO₂ atmosphere. Cells on the lower surface were counted in the same way as the cell migration assay.