2.7.3 MPO-DNA Sandwich-ELISA

NET-specific MPO-DNA complexes were measured as described elsewhere (49). NET samples and NET standards were diluted 1:50 and 1:20 in PBS with 2.5 mM EGTA (Sigma-Aldrich, Saint Louis, MO, USA), respectively. The anti-MPO capture antibody (ab267425, abcam, Cambridge, UK) was diluted 1:250. Blocking was performed using PBS containing 5% BSA (Sigma-Aldrich, Saint Louis, MO, USA) for 2 h at room temperature. Overnight incubation of samples was performed at 4°C on an orbital shaker at 25 rpm. The Anti-DNA Peroxidase detection antibody (Cell Death Detection ELISA^PLUS, 11774425001, Sigma-Aldrich, Saint Louis, MO, USA) was diluted 1:500. Then, 15 minutes after addition of tetramethylbenzidine (TMB, Sigma-Aldrich, Saint Louis, MO, USA), the reaction was stopped by adding 2 M H₂SO₄. The absorbance was measured at 450 nm with a microplate reader (Flex Station^® 3, Molecular Devices, San Jose, CA, USA). The number of MPO-DNA complexes was determined relative to the NET standard curve, which was created by mixing nuclease-digested NET supernatants of PMA-stimulated neutrophils from 5 different donors, as described before (49), and subsequently diluting this mix 1:2 afterwards. NET samples were freshly thawed before every measurement. For each treatment, the delta of expelled MPO-DNA after PMA stimulus and accumulated MPO-DNA without PMA stimulus is displayed to eliminate the background produced by cell death.