Transwell Assay for Cell Migration and Invasion

The effect of AR down-modulation on E006AA cell migration and invasion was performed using 8-µm transwell filters (Corning) with minor modification as described previously 21 (link). For the invasion assay, the upper compartment was coated with 50 µg Matrigel (BD Biosciences) to form a matrix barrier. A suspension of cells (4 × 10⁴ in 200 µl) in basal medium containing 0.1% BSA was added to the upper compartment. The lower compartment was filled with 400 µl basal medium containing 1% FBS as a chemoattractant. After 24 h, 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 in ten randomly selected fields (at 40X magnification). The experiment was performed in quadruplicates and repeated at least three times independently.