Mab699

Migration and invasion studies were performed using BD Biocoat migration and Matrigel invasion chambers (Becton-Dickson Labware), respectively. Serum free DMEM was added to bottom and top chamber of inserts and allowed to hydrate for 2 h at 37°C with 5% CO₂. Next, 0.5 × 10⁵ cells were added to the top chamber of inserts and 100 ng/ml CXCL16 (Peprotech, NJ) was added as chemo-attractant in the bottom chamber. To determine if the migration and invasion of LuCa cells is mediated specifically by CXCR6-CXCL16 interaction, cells pre-incubated with 1.0 μg/ml anti-CXCR6 antibody (MAB699, R&D Systems) were added to the top chamber in one well of Matrigel or control inserts and allowed to migrate/invade under chemotactic gradient of CXCL16 for overnight at 37°C and 5% CO₂. After incubation, non-migrating cells on the upper surface of the membrane were removed with a cotton swab. Cells at the bottom surface of the insert were fixed with 100% methanol for 2 min, stained for 2 min with crystal violet (Fisher Scientific), and rinsed twice with de-ionized water. Migrated/invaded cells were counted by microscopy at 40× magnification. All experiments were repeated three times to validate the results.