8 μm pore transwell filters

Cells were harvested after serum-free starvation for 12 hrs, and were resuspended in plain DMEM media. Ten thousand cells were applied to 8-μm pore transwell filters (Corning). DMEM media containing 10% FBS were added to the bottom chamber as attractants. After incubation for 24 hrs, the nonmigrated cells at the top of the filter were removed and the migrated cells at the bottom of the filter were fixed with 4% paraformaldehyde and were stained with colloidal staining method. The number of migrating cells in each chamber was quantified by counting nine randomly chosen fields under ×20 magnification using a bright-field microscope. Each condition was performed in duplicate, and the average number of cells per field was represented. Experiments were repeated three times.

Cells were collected and resuspended in FBS-free DMEM. Twenty thousand cells were placed on 8-μm pore transwell filters (Corning, USA). For invasion assays, filters were coated with Matrigel (BD, UAS) in advance and for migration assays, this step was omitted. DMEM with 10% FBS was added to the bottom chamber as an attractant. Following incubation for 24–36 h, nonmigrated cells at the top of the filter were removed and cells at the bottom of the filters were fixed with 4% paraformaldehyde and stained with crystal violet. The total number of invaded cells in each chamber were quantified by counting at least four randomly chosen fields under ×20 magnification using a bright field microscope (IX71; Olympus).