Cxcl12

Cell migration assay was performed using a 96-well plate based chemotaxis system (Neuroprobe, Gaithersburg, MD), as described previously [27 (link)], in three independent experiments. CD4⁺ cells were isolated from mouse spleen cells using magnetic bead negative selection (Stemcell® Mouse CD4⁺ T Cell Enrichment Kit), were pre-activated with anti-CD3 and anti-CD28 antibodies (eBioscience) for 3 h in RPMI medium containing 10 % fetal calf serum and resuspended at 1 × 10⁷ cells/ml. Lower chambers were loaded with 29 μl of the chemokine CXCL12 in the indicated concentration (ranging from 1 μg/ml to 0.01 ng/ml; ImmunoTools, Friesoythe, Germany) and overlaid with a 5-μm pore-size filter membrane. The cells (20 μl, i.e. 2 × 10⁵ cells) were loaded on the hydrophobic surface surrounding each well on the membrane (upper chamber). CGS21680 was added in different concentrations in order to assess its effect on migration (upper and lower chambers always containing the same CGS21680 concentration). After 3 h of incubation, cells in the lower chamber were transferred to a 96-well plate and counted automatically using a Becton Dickinson FACSCanto II with a 96-well high throughput sampler (HTS) device.

Recombinant human chemokines and growth factors were from Pepro Tech (Rocky Hill, N.J., USA; CXCL11) or Immunotools (Friesoythe, Germany; CXCL12, epidermal growth factor). Staurosporine was purchased from Calbiochem (Merck-Calbiochem, Darmstadt, Germany). The non-peptide antagonist AMD3100 (= plerixafor, Mozobil, selective for CXCR4) was obtained from Sigma-Aldrich (St. Louis, Mo., USA) and CCX733 (selective for CXCR7) was a generous gift from Mark E.T. Penfold from ChemoCentryx (Mountain View, Calif., USA). Staurosporine and antagonists were dissolved in dimethylsulfoxide and diluted from these stock solutions.

5 × 10⁵ cells were cultured on the top chamber of a transwell culture insert (Corning, pore size 5 μm) and were allowed to migrate toward media containing 100 ng/ml CXCL12 (ImmunoTools) for 16 h. The total cell number in the lower chamber was determined using hematocytometer.