Quantifying Cell Migration and Invasion

In wound scratch assay, 1.7 × 10⁵ cells were used in a 24-well plate. Quiescent cells were wounded [16 (link)] and left un-stimulated or stimulated for 28 h, in the absence or presence of the indicated compounds. To avoid cell proliferation, cytosine arabinoside (Sigma-Aldrich) at 50 μM (final concentration) was included in the cell medium. Different fields were analyzed using DMIRB inverted microscope (Leica) equipped with N-Plan 10× objective (Leica). Phase-contrast images, representative from three different experiments, were captured using a DFC 450C camera (Leica) and acquired using Leica Suite Software (Leica). The wound width was calculated using Image J Software and expressed as % of the decrease in the wound area. Migration and invasion assays were performed by using collagen- or Matrigel- pre-coated Transwells with 8 μm polycarbonate membrane (Corning; Corning, NY, USA), respectively [16 (link)]. The indicated compounds were added and cytosine arabinoside was included in cell medium. Cells were allowed to migrate or invade for seven or 18 h for LNCaP cells respectively and for nine h or 24 h for DuCaP and 22Rv1, respectively. Migrating or invading cells were then scored [16 (link)].