Quantification of Neutrophil Mediators

Concentration of human MPO in PMN supernatants was quantitated by commercial ELISA kit (R&D Systems, Minneapolis, MN, USA) as previously described [19 (link), 41 (link)]. Human neutrophil elastase release was assessed by sandwich ELISA: diluted supernatants were applied to 96-well high binding microloan ELISA plates (Greiner bio-one, Germany) pre-coated overnight with anti-human neutrophil elastase rabbit polyclonal antibody (1:500 in PBS, Calbiochem, 481001, EMD Millipore, MA, USA). After blocking with 1% BSA for 1 hr, a secondary anti-human neutrophil elastase antibody was applied (1:2000 in PBS, IgG1, cat #: MA1-10608, ThermoScientific, Hudson, NH) followed by the addition of a horse radish peroxidase-linked (donkey) anti-mouse IgG antibody (1:2000 in PBS, NA934V, GE Healthcare, UK) for 1 hr at room temperature. Blue coloration developed in the presence of the Pierce TMB Substrate Kit (ThermoFisher Scientific, Waltham, MA, USA), and results were quantitated using a human neutrophil elastase standard.