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.