Figure 5.

PA-dPEG24 inhibition of PMA-initiated neutrophil extracellular trap (NET) formation with human neutrophils (PMN). (A) PA-dPEG24 inhibition of PMA-initiated NET formation assayed by fluorescence microscopy for DNA (DAPI), neutrophil elastase (αNE), and histone H3 (αhistone). The first row shows unstimulated neutrophils, the second row shows neutrophils stimulated with PMA and hydrogen peroxide (H₂O₂), and third row shows neutrophils stimulated with PMA + H₂O₂ in the presence of 5 mM PA-dPEG24 peptide inhibitor of complement C1. The first columns are slides probed with DAPI to visualize DNA, the second columns are slides probed with anti-αNE antibody, and the third row is probed with anti-histone H3 antibody. Representative images are shown. (B) Oxidized PA-dPEG24 [ox peptide inhibitor of complement C1 (PIC1)] demonstrates no activity in the PMA-initiated NET assay by measuring free DNA with PicoGreen. Data are means of (n = 3) independent experiments ± SEM. (C) Oxidation of the sulfhydryl groups of PA-dPEG24 (oxPIC1) demonstrated with Ellman’s reagent. Data are means of (n = 3) independent experiments ± SEM.