Quantifying Neutrophil Extracellular Traps

NET-production of peripheral blood neutrophils was determined in vitro as previously described [30 (link)]. Briefly, freshly isolated neutrophils were plated on coverslips and treated with 100 nM PMA (phorbol-12-myristate-13-acetate, Sigma Aldrich) in RPMI containing 0.05% human serum albumin (HSA, Gibco® ThermoFisher) or left untreated. After fixation with 2% PFA (paraformaldehyde) at 2 and 4h, the coverslips were washed and permeabilized with 0.5% TritonX-100/PBS for 5 min. Subsequently, cells were incubated for 2h with an anti-chromatin antibody (PL2-3, produced in house), a rabbit-anti-neutrophil-elastase-antibody (Calbiochem, Merck). After washing, goat anti-mouse Alexa568, goat anti-rabbit Alexa488 and Hoechst 3342 (all ThermoFisher) were added and incubated for 1 hour on ice. After several washing steps, immunofluorescence microscopy was performed using the Evos FL Auto 2 fluorescent microscope from Invitrogen. Images were analysed using ImageJ as described before. Briefly using the settings and thresholds described by Brinkmann et al. [30 (link)] the NET-rate was calculated as: NET-rate = 100*Objects counted (chromatin channel)/Objects counted (Hoechst channel).