Cell Migration and Invasion Assays

Cell migration assays were performed using 6.5 mm Transwell with 8.0 μm pore polycarbonate membrane insert (Corning) as previously described [9 (link)] with some modifications. In brief, cells (1.0 × 10⁵) in 200 μl serum-free RPMI-1640 medium were loaded into the top chambers, and 600 μl RPMI-1640 media containing 10% FBS was added into the lower chamber as a chemoattractant. After incubation for forty-eight hours at 37°C in 5% CO₂, the non-migratory cells on the upper surface were removed by a cotton swab and the cells that had migrated to the other side of the membrane were fixed with 4% paraformaldehyde for 20 minutes and stained with Wright-Giemsa. Values for migration were obtained by counting cells in 10 fields (2 × (centre + 4 quadrants)) per membrane (× 20 objective) and averaged for three wells. For cell invasion assays, cells (1.0 × 10⁵) were suspended in 200 μl serum-free RPMI-1640 and seeded on the matrigel-coated membrane in each insert of biocoat matrigel invasion chambers with 8.0 μm PET membrane (Corning). Forty-eight hours later, the cells that had invaded the matrigel and moved to the other side of the membrane were fixed, stained and counted as in the migration assay. The experiment was repeated three times independently.