Transwell Assay for Cell Migration and Invasion

The effect of AR silencing on cell migration was performed using 8-μm transwell filters (Costar, Corning, NY) with modification as described previously 39 (link). For the invasion assay, the upper compartment was coated with 50 μg Matrigel (BD Biosciences, San Jose, CA) to form a matrix barrier. A suspension of cells (20 × 10⁴ per filter for migration and 40 x 10⁴ per filter for invasion) in medium containing 1% FBS and 0.1% BSA was added to the upper compartment. The lower compartment was filled with 400 μl basal medium containing 10% FBS as chemoattractant. After 24 h for migration or 48 h for invasion, the non-migratory cells on the upper surface were removed by a cotton swab and the cells on the lower surface were fixed and stained with the Diff-Quick solution (Dade Behring, Deerfield, Illinois). Migrated or invaded cells in each transwell filter were counted from ten randomly chosen fields. The experiment was performed in quadruplicate and repeated three times independently.