Transwell invasion assay

Cal27 (2×104 cells/0.4 ml) cells were seeded in the upper chamber of the Transwell inserts (8-µm pore size) pre-coated with Matrigel (both from Corning, Corning, NY, USA) and exposed to FBS-free medium with or without 10 µM nicotine. Medium containing 10% FBS was placed in the lower chamber, and cells for each treatment were incubated for 24 h at 37°C in a humidified atmosphere with 95% air and 5% CO₂. Then, the non-invasive cells in the upper chamber were removed with a cotton swab, and the invaded cells were fixed with 4% formaldehyde for 15 min and then stained with 0.1% crystal violet in 0.01 M PBS for 15 min after being washed with PBS. The number of cells that penetrated the membrane was counted, and images were captured under a light microscope at a magnification of ×200, as previously described (21).