MPO is required for NET formation. Neutrophils were activated with PMA for 4-6 hours to stimulate NET formation, then stained with the DNA dye Sytox green (A-C) or (D) immunostained with antibodies against the histone H2A/H2B/DNA complex (red) and neutrophil elastase (green). (A) Neutrophils from a control donor formed NETs after PMA stimulation, and naive cells had small, condensed nuclei. (B) Neutrophils from completely MPO-deficient donors did not form NETs, as indicated by their condensed nuclei similar to those of unstimulated neutrophils. (C) Partially MPO-deficient neutrophils made NETs. (D) Extracellular DNA released from control neutrophils stained with antibodies against chromatin and neutrophil elastase, 2 known NET components. In completely MPO-deficient neutrophils, as in naive neutrophils,20 neutrophil elastase was localized in granules and chromatin remained condensed. Experiments with CD neutrophils were repeated at least 6 times with 3 independent, unrelated donors. Experiments with PD neutrophils were repeated at least 3 times with 3 independent, unrelated donors. Scale bar, 25 μm.
