Trans well

Cells were suspended in RPMI 1640 medium and placed in the upper compartment of an 8 μm Transwell^® (3.2 mm diameter; Neuro Probe, Gaithersburg, MD, USA). The lower compartment was filled with RPMI 1640 medium supplemented with FBS. After 24 h, the filter was washed with PBS and the migrated cells on the filter membrane were stained using a Diff-Quik Stain Kit (Sysmex, Tokyo, Japan). For invasion assays, the upper compartment of an 8 μm Transwell^® (6.5 mm diameter; Costar, Cambridge, MA, USA) was coated with Matrigel^® (1 mg/ml) before starting the assay. Each assay was conducted at least three times, and three random fields using 20 × magnification were analyzed for each filter membrane (21 (link)).

Migration assays were performed in a transwell (Neuro Probe, Inc., Gaithersburg, MD, USA) coated with 10 μg/mL fibronectin. A549 cells (1×10⁶ cells/mL) suspended in serum-free medium were added to the upper chamber of the transwell inserts. To the lower chamber, medium containing 3% FBS was added. After 5 h incubation, non-migrated A549 cells were scraped off the upper surface of the membrane; the migrated A549 cells on the lower surface were stained by Diff-quick and counted under four randomly chosen high-power fields (20× magnification). All of the experiments were repeated at least three times with two replicates each.