Uncoated transwell chambers

Uncoated transwell chambers and modified Boyden chambers (BD Biosciences) with 8 µm pore size were used to examine the cell migration and invasion ability respectively. In brief, C4-2 or PC3M cells were suspended in serum-free medium and were seeded to the upper chambers. The medium containing 5% fetal bovine serum was added to the lower chambers. The cells and chambers were incubated at 37 °C for 22 hours. After incubation, cells that were adhered to the upper surface of the chamber were wiped off. The cells on the lower surface were fixed, stained with crystal violet and counted under a light microscope.

Migration and invasion of cells were determined using Transwell analysis. Cell invasion was measured using Matrigel-coated Transwell chambers (BD Biosciences, Franklin Lakes, NJ, USA), and cell migration was measured using uncoated Transwell chambers (BD Biosciences). Invasion assay was performed using Transwell inserts pre-coated with Matrigel (BD Biosciences). RPMI-1640 medium supplemented with 12% FBS was added to the lower chamber. The treated cells were digested with trypsin and a cell (2×10⁵ cells/ml) suspension was incubated in the upper chamber at 37°C for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min and stained with 0.1% crystal violet for 20 min at room temperature. The stained cells were observed under a light microscope (magnification, ×200).