2.9. Transwell Migration Assay and Invasion Assay

The invasion and migration of pancreatic cancer cells were tested by using 8 μm transwell chambers (Millipore Corporation, Burlington, MA, USA). For invasion assay, the upper chambers were coated with 1 : 8 diluted Matrigel and incubated at 37°C for at least 1 h. PANC-1 (4 × 10⁴) and BxPC-3 cells (4 × 10⁴) in different groups were starved in 200 μL serum-free medium in the upper chamber with (invasion) or without (migration) Matrigel (Corning, No. 356234). The lower chambers were filled with 600 μL culture medium containing 10% FBS, which act as chemoattractant. After incubation at 37°C for 24 h even 48 h, the cells were stained with 4% paraformaldehyde and 0.5% crystal violet solution for 30 min. Finally, the number of cells that migrated or invaded w counted in five selected areas under 100x magnification using an inverted microscope. Each result was repeated three times.