8 m pore chambers

Cell invasiveness and migration were measured a Transwell assay using 8 µm pore-chambers (Corning, USA). Briefly, a 2.5 × 10⁵ cells/mL suspension was generated from treated cells using serum-free DMEM medium. Two hundred microliters (200 μL) cell suspension and 600 µL DMEM medium containing 10% FBS were added into the upper and lower chamber, respectively. After 24 h of incubation, the cells in the chamber were fixed and stained with 100% paraformaldehyde and 0.5% crystal violet. The cells in the upper chamber were removed and evaluated under a microscope. In order to further evaluate cell invasiveness, the assay was repeated in a Transwell assay with Matrigel (BD Biosciences, Bedford, MA, USA).

Cell migration was measured by the transwell method, using 8-µm pore chambers (Corning, NY, cat # 3244). Briefly, different numbers of cells were seeded onto the upper chamber in serum-free medium. The lower chamber was filled with 600 µL of medium containing 1 µM KA (or not, in the control group). After 24 hours of incubation, the cells of the upper chamber were gently removed with cotton swabs, and the lower side of the membrane was dipped in 95% methanol and stained with crystal violet for 30 minutes. Five random fields were counted under a microscope.