Isotype control mouse igg1

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).

NETs were induced in peripheral blood neutrophils by PMA with or without anti-MYL6 antibody as above. Before and 30 min, 1 h, and 3 h after incubation, the samples were fixed with 4% paraformaldehyde for 15 min and permeated with 0.5% Triton X-100 for 5 min at room temperature (RT). Thereafter, the samples were reacted with 1:100 dilution of anti-β-actin monoclonal antibody (mAb; mAbcam 8226, mouse IgG1; Abcam) or equivalent concentrations of isotype control mouse IgG1 (Abcam) at 4°C overnight. After rinsing with PBS, the samples were reacted with 4 μg/mL Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody (Abcam) and 100 nM Acti-stain 555 phalloidin (Cytoskeleton, Denver, CO, USA) for 1 h at RT in the dark. In cells, actin exists in two different forms: nonpolymerized granular form (G-actin) and polymerized filamentous form (F-actin). Anti-β-actin antibody recognizes G-actin [18 (link)], whereas phalloidin binds specifically to F-actin. The samples were finally mounted with the mounting solution containing DAPI. This preliminary assay revealed that G-actin polymerization into F-actin occurred in early during NET formation (0–30 min after PMA stimulation), followed by F-actin degradation (Fig. S1). These findings were consistent with previous reports of Stojkov et al. [15 (link)] and Metzler et al. [16 (link)].