Matrigel coated transwell insert chambers

Migration and invasion assays were performed as previously described (25 (link)). ACHN and 786-O cells had been transfected with si-RAC2 or si-NC 48 h before subsequent experimentation. Prior to the migration and invasion assays, cells were incubated in DMEM without serum for 6-8 h. Boyden Transwell chambers and 24-well plates (Corning Inc.) with 8-µm membrane filters were used in the migration and invasion assays. Serum-starved cells (1×10⁵) were seeded into the upper chambers in serum-free medium, and the lower chambers were filled with DMEM containing 10% FBS. After incubation for 24 h at 37°C, the lower chamber was washed twice with PBS and fixed with 100% methanol for 15 min at room temperature, and stained with 0.1% crystal violet dye for 20 min at room temperature. Following washing of the chamber again three times with PBS, non-migrated and non-invaded cells were removed from the upper chamber with a cotton bud. Migrated cells in lower chambers were observed in five randomly selected fields under a light microscope (Olympus CX41-32C02; Olympus Corporation) at 100x magnification. Based on the migration assay, a cell invasion assay was performed in Matrigel-coated Transwell insert chambers (BD Biosciences), which had already been incubated at 37°C for 6-8 h. The remaining procedure was performed as described for the cell migration assays.