Cxcl12 sdf 1

For invasion assays, MDA-MB-231 cells transfected with 0.25 μg of luciferase expression cassettes and cotransfected with CXCR4 siRNA or control siRNA for 48 h were reseeded onto the upper chambers of 24-well invasion inserts of 8 μm pore-sized membranes (BD Biosciences) in serum-free DMEM at a density of 3 × 10⁵ cells per well and incubated for 24 h. Six hundred microliters of serum-free DMEM + 0.1% bovine serum albumin or serum-free medium + 10 nM CXCL12/SDF1 (R&D Systems, Minneapolis, MN) was used as the chemoattractant in the lower chambers. The membranes were removed from the inserts and incubated with luciferase lysis buffer (Promega, Madison, WI) for 15min, and then luciferase activity was measured according to the manufacturer’s protocol. For migration experiments, MDA-MB-231 cells were seeded at the same density as for invasion assays onto the upper chamber of 24-well migration plates and 600 μl of serum-free DMEM + 0.1% bovine serum albumin, or serum-free medium + CXCL12 was added to the lower chambers of migration plates and incubated for 24 h. Cell-Titer-Glo reagent (Promega) was then directly added to the lower chamber to measure luminescence associated with the migrating cells. For both invasion and migration assays, a ZENYTH 3100 reader (Anthos Labtec) was used to measure luciferase activity or luminescence, respectively.