Quantifying Plasma Neutrophil Extracellular Traps

The amount of plasma NETs was addressed by ELISA, as previously described (18 (link)). Briefly, high binding 96 well plates were coated overnight at 4°C with mouse anti human neutrophil elastase (NE) 1:2000 (Cat 481001, Calbiochem, Darmstadt, Germany) in coating buffer from the cell death detection ELISA kit (Roche, Basilea, Switzerland). We washed the plates three times with PBS/Tween20 and blocked the non-specific binding sites with 1% bovine serum albumin (BSA) in PBS for 6 hrs at room temperature for the detection of DNA-NE complexes as previously described (18 (link)). The plasma samples were diluted 1:10 in 1% BSA and incubated overnight at 4°C. After washing three times with PBS/Tween 20, we incubated the plates with the anti-human DNA-POD antibody from the cell death detection ELISA kit (Roche, Basilea, Switzerland). We washed the plate five times with PBS/Tween 20 and applied the TMB substrate (Thermofisher Scientific, Massachusetts, USA). The plate was read at 450 nm after applying stop solution and the optic density index (ODI) was calculated as previously described (18 (link)).