Cell migration and invasion assay

Cell migration and invasion was measured in a Boyden chamber system according to standard protocols [19]. MDA-MB-231 and 4T1 cells were cultured in serum-free medium for 24 h. For migration assays, cells (1 × 10⁵) were placed in the upper chamber with non-coated membrane (24-well insert; 8-μm pore size; Corning Inc.). For invasion assays, cells (1 × 10⁵) were placed in the top chamber with Matrigel-coated membrane (24-well insert; 8-μm pore size; Corning Inc.) In both assays, cells were plated in 0.2 ml serum-free medium in the top chamber, and the lower chamber was loaded with 0.5 ml medium containing 10% FBS. The total number of cells that migrated into the lower chamber was counted after 16 h of incubation at 37 °C with 5% CO₂. Cells that had not penetrated the filter were wiped out with cotton swabs, and cells that had migrated to the lower surface of the filter were stained with 0.5% crystal violet, examined by bright field microscopy, and photographed. Crystal violet was then dissolved with ethanol, and absorbance was read at 570 nm. Values for migration/invasion were expressed as the average number of optical density (OD)₅₇₀ per assay.