Mouse anti human cd177 antibody

Recombinant PECAM-1, junctional adhesion molecule-1 (JAM-1), and CD177 were purchased from Sino Biological Inc. (Beijing, China). Fluorochrome dihydrorhodamine (DHR), phorbol myristate acetate (PMA), and normal human immunoglobulin (Ig)G were purchased from Sigma (St Louis, USA). For indirect enzyme-linked immunosorbent assay (ELISA), mouse anti-human CD177 antibody was purchased from Sino Biological Inc. and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was purchased from Abcam (Cambridge, UK). For flow cytometry analysis, phycoerythrin (PE)-conjugated mouse monoclonal antibody against human CD177 and the isotype control mouse IgG1 were purchased from BioLegend (San Diego, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal antibody against human PR3 and the isotype control mouse IgG1 were purchased from Abcam. For magnetic neutrophil sorting, anti-PE microbeads and separation columns were purchased from Miltenyi Biotech (Bergisch-Gladbach, Germany). For Western blot, antibodies against SHP-1 (C14H6) and phosphor-SHP-1 (Tyr564) were purchased from Cell Signaling Technology (Boston, MA, USA), and mouse anti-human GAPDH antibody was purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

The interaction between CD177 and PECAM-1 was detected by ELISA with recombinant soluble PECAM-1 (sPECAM-1) at 2 μg/ml as the solid-phase antigen. sPECAM-1 in a coating buffer (0.05 M bicarbonate buffer, pH 9.6) was used to coat the wells of half of a polystyrene microtiter plate (Nunc-Immuno plate; Nunc, Roskilde, Denmark) and was incubated overnight at 4 °C. The other half of the plate was coated with coating buffer alone to establish antigen-free wells. The wells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and incubated with CD177 at various concentrations for 1 h at 37 °C. After washing three times with PBS-tween 20 (PBS-T), the wells were incubated with mouse anti-human CD177 antibody (1:1000; Sino Biological) for 1 h at 37 °C, and HRP-conjugated goat anti-mouse IgG (1:1000) was used as the secondary antibody. Tetramethylbenzidine (TMB; Sigma, St. Louis, MO) was used as the substrate and the reaction was stopped by the addition of sulfuric acid 0.5 mol/L (Carl Roth GmbH, Germany). Optical densities of formed complexes were measured at 450 nm using a microplate reader (Bio-Rad, Tokyo, Japan).