4.6. Invasion Assay

Invasion assays were done in transwell invasion chambers with an 8 μm pore size filter membrane (Corning, ME, USA). The cells were treated with FDI-6 before the experiment 24 h prior. The inserts were coated with 100 µL of 0.3 mg/mL of Matrigel (Corning, Kennebunk, ME, USA) and were incubated at 37 °C overnight. After the Matrigel layer formed inside the inserts, the cells were pre-treated with mitomycin C (10 µg/mL) for 2 h and were then transferred into each insert as 6.5 × 10⁴ cells/well in 200 μL of DMEM supplemented with 1% FBS. The lower chambers were added with 600 μL of DMEM supplemented with 10% FBS. The inserts were then merged with the lower chambers and were incubated at 37 °C for 16 h. After incubation, the cells that did not migrate through the pores were removed with cotton swabs. The cells on the underside of the inserts were fixed with 25% (v/v) methanol for 15 min and then stained with 1% (w/v) crystal violet in 25% (v/v) methanol for 15 min. The stained cells were washed with distilled water for 30 s. The inserts were dried at room temperature overnight. The images of the stained cells on the underside were obtained by an inverted microscope (Olympus, Tokyo, Japan). The average number of migrated cells were calculated from five randomly chosen fields from each insert.