2.3 NETs measurements
DNA-NETs formed by neutrophils adherent to fibrinogen-coated surfaces were visualized by confocal microscopy after DNA staining with DRAQ-5 and FITC-conjugated anti-myeloperoxidase antibody (Abcam, Cambridge, UK) (Totani et al., 2016). Extracellular DNA was quantified by Quant-iTTM dsDNA high-sensitivity assay kit (Molecular Probes by Life Technologies, Oregon, USA) as previously described (Totani et al., 2016). Briefly, 2.5µl of endonuclease (Nuclease micrococcal, from Staphylococcus aureus 50 U/ml) (Sigma-Aldrich, Milan, Italy) were added to cell samples (500 µl) and incubated at 37°C for 10 min, followed by 5 µl of EDTA (0.5 M) to stop the reaction. Samples were centrifuged at 10.000 x g in an Eppendorf centrifuge, for 3 min and supernatants stored at -20°C until DNA quantification.