Transwell chambers with 8.0 μm pore polycarbonate membrane insert

Transwell chambers with 8.0 μM pore polycarbonate membrane insert (Corning, USA) assessed cell migration and invasion abilities. For cell invasion assay, 40 μL matrigel solution (Matrigel: medium = 1: 4) was added to transwell inserts and solidified for 3 h at 37 °C. Then 500 μL DMEM with 10% FBS was added to the lower chamber. Cells were resuspended with serum-free medium and plated into transwell inserts at 1 × 10⁵ cells/well. The cells on the filter’s upper surface were removed after they were cultured at 37 °C for 24–72 h. For cell migration assay, the matrigel solution was not needed on the inserts, and other steps were as same as invasion assay. Cells on the lower surface of the filter were fixed with 4% paraformaldehyde for 20 mins and then washed twice with PBS before they were stained with 1% crystal violet solution for 10 mins. The stained cells were counted by a microscope.

The migratory ability of cells was evaluated using 6.5 mm Transwell chambers with 8.0 μm pore polycarbonate membrane insert (Corning, NY, USA). Cells prepared in FBS-free medium were seeded onto the upper chambers, and medium with 10 % FBS was added to the bottom chambers as a chemoattractant. cDDP was given to the cells 30 min later. After 12 h, migrated cells located on the lower surface of chambers were fixed and stained with crystal violet. The cells were photographed using an inverted microscope and counted in 10 randomly selected fields at a 200× magnification using Image Pro-Plus software 6.0 (IPP 6.0, Media Cybernetics, MD, USA). The assay was carried out three times.