MMP9 + Isotype

1. (a) (b) (c) (d) MMP9 + anti MMP9 MMP9 + Isotype Supplementary Figure 5: Antibodies for VEGF, IL-8, ENA-78 and MMP9 used in cell culture experiments (figure 4e) were tested for their neutralizing activity. A strong and almost complete inhibition was found for all tested antibodies [(a) VEGF; (b) IL-8; (c) ENA-78; (d) MMP9]. Supplementary Figure 5 Methods (a) Human umbilical vein endothelial cells (HUVEC) were treated with 5 ng/ml of recombinant human VEGF together with 2 µg/ml of polyclonal goat anti human VEGF antibodies (AF-293-NA, R&D Systems, USA) or isotype control. 48 hours after treatment a proliferation-assay (EZ4U, Biomedica, Vienna Austria) was performed according to the manufacturer's instructions. (b,c) Human granulocytes were obtained by lysis of red blood cells in whole blood of 4 healthy probands followed by selective separation of CD66 positive cells using a MACS MicroBead Kit (Miltenyi Biotec, Germany). Granulocytes were stimulated with 5 ng/ml recombinant human IL-8 or 5 ng/ml ENA-78. For inhibition 2 µg/ml monoclonal mouse anti human IL-8 antibodies (AF-293-NA, R&D Systems, USA), 2 µg/ml goat anti human ENA-78 antibodies (AB-254-PB, R&D Systems, USA) or isotype control antibodies were added. Myeloperoxidase secretion was measured by ELISA (DMYE00, R&D Systems, USA). (d) For MMP9 inhibition an enzyme assay for MMP-activity was used. A specific MMP-substrate was incubated (Bachem, Basel, Switzerland) for 1 hour with 5 ng/ml recombinant MMP9 (R&D) together with 2 µg/ml MMP9 antibody or isotype control. The emitted fluorescence was measured with a fluorometer (Fluostar Optima, BMG Labtech, Vienna, Austria).