Plasma NETs were measured using an ELISA-based assay. Briefly, an anti-MPO antibody (Santa Cruz Biotechnology, sc-390109) was coated on a 96-well plate followed by blocking the non-specific binding sites with 5% BSA. 15 μl of plasma and 35 μl of PBS were added into each well and incubated for 2 hours at room temperature under shaking conditions. After washing, the attached MPO-DNA complexes in the wells were quantified by an anti-DNA antibody conjugated with peroxidase using a commercial ELISA kit (Roche, Cat#11774425001).
To evaluate NET release in vitro, the endothelial cell monolayer was primed with TNF-α (100 ng/ml), to which neutrophils and platelets isolated from mice were added either separately or jointly with a ratio of 1:25, with or without the indicated agonist and antibody, and incubated for 2 hours at 37°C. After incubation, cells were treated with micrococcal nuclease to detach the released NETs from neutrophils, and NETs were further quantified using the Sytox green assay [25 (link)].
To evaluate NET release in vitro, the endothelial cell monolayer was primed with TNF-α (100 ng/ml), to which neutrophils and platelets isolated from mice were added either separately or jointly with a ratio of 1:25, with or without the indicated agonist and antibody, and incubated for 2 hours at 37°C. After incubation, cells were treated with micrococcal nuclease to detach the released NETs from neutrophils, and NETs were further quantified using the Sytox green assay [25 (link)].
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