AT inhibits HRPII activation of neutrophils and NET formation. (A) Human blood neutrophils were isolated and treated with HRPII (100 nM) in absence or presence of AT for 4 hours. Cells were then fixed in 4% paraformaldehyde, permeabilized with 0.2% TritonX-100/PBS, and stained with anti-neutrophil elastase (goat polyclonal) antibody, anti-citrullinated H3 (rabbit polyclonal) followed by Alexa Fluor-488 conjugated anti-goat and Alexa Fluor 555-conjugated anti-rabbit antibody. DNA was stained with DAPI. Quantification of NET forming cells and citrullinated H3-positive cells per field represented as the percentage of the total cells. (B) The same as panels above except that human blood neutrophils were fixed and stained with anti-PAD4 (rabbit polyclonal) and antihistone (H3) (mouse monoclonal) antibody followed by Alexa Fluor 555-conjugated anti-rabbit antibody and Alexa Fluor 488-conjugated anti-mouse antibody. DAPI was used to stain the nucleus. Quantification of PAD4 nuclear translocation is represented next to the panel. (C) Human blood neutrophils were treated with HRPII in the absence and presence of AT. PAD4 levels in nuclear extract were analyzed through western blot. PCNA was used as loading control. Densitometric analysis is presented. (D) Human blood neutrophils were treated with HRPII in the absence and presence of AT. TNF-α levels in supernatant of neutrophils were measured using commercial ELISA.