2.6. Transwell migration and invasion assays

The changes in cell migration were assessed in 24‐well plates using the Transwell system (Corning, NY). Cells were treated with mitomycin C (10 μg/mL) for 3 hours and washed 3 times with PBS to inhibit cell proliferation. The Transwell inserts (6.5 mm diameter; 8 μm pore size) were incubated in DMEM at 37°C for 1 hour prior to use. Following this, 600 μL of DMEM (20% FBS) was added to the outer wells of the Transwell plate and the Transwell inserts placed inside. A total of 1 × 10⁵ cells in DMEM (10% FBS) were seeded onto the Transwell insert. The plate was incubated at 37°C for 24 hours. Following this, the cells that had migrated through to the bottom of the outside well, using the higher FBS concentration as a chemoattractant, were manually counted. Triplicate wells were seeded for each experimental condition. The Transwell invasion assay was performed according to this protocol with the exception that 2 × 10⁵ cells were seeded onto a Transwell insert coated in Matrigel reduced growth factor at 0.3 mg/mL (Corning, NY) and cells allowed to migrate for 48 hours prior to counting.