Transwell migration and invasion assays

The migratory ability of NSCLC cells was determined using Transwell chambers (8 µm; BD Biosciences). The Transwell invasion assay was performed with experimental steps that were similar to the migration assay, except that Matrigel (BD Biosciences) was used to coat the chambers. In brief, 100 µl of FBS-free culture medium containing 5×10⁴ cells were added into the upper compartments, while the lower compartments were covered with culture medium supplemented with 20% FBS, which was used as a chemoattractant. Following a 24-h incubation, the non-migrated and non-invaded cells were gently removed with a cotton swab, whereas the migrated and invaded cells were fixed with 4% paraformaldehyde at room temperature for 20 min and stained with 0.1% crystal violet at room temperature for 20 min. An inverted light microscope (×200 magnification; Olympus Corporation) was used to image and count the number of migrated and invaded cells.