Fig. 3.

ATP and ADP participate in neutrophil activation but not in NETs formation. (A) Representative images of PAF-activated (Left Panel) and PMA-activated neutrophils (Right Panel). Purified neutrophils were primed with TNF-alpha and were respectively activated with PAF or PMA for 3 h. After NETosis induction, neutrophils were immunostained with anti-CitH3 antibody (in red) or anti-NE antibody (in green) and Hoechst 33342 for DNA labeling (in gray for human neutrophils or blue for mouse neutrophils). (B) Representative images of CitH3 expression on mouse neutrophils. Purified neutrophils were primed or not with TNF-alpha excepted and activated or not with ATP, ADP, or PAF. Neutrophils were immunostained with anti-CitH3 antibody (in red) and Hoechst 33342 for DNA labeling (in gray). Colocalization of both labeling (DNA/CitH3+) was represented in pink. (C) Graph depicts the percentage of NETs formation from mouse neutrophils in each condition (mean ^+/− SEM). PAF-activated neutrophils condition was used as positive control corresponding to 100% of NETs formation. (D) Representative images of NE expression on human neutrophils. Human purified neutrophils were primed or not with TNF-alpha and activated or not with ATP, ADP, or PAF. Neutrophils were immunostained with anti-NE antibody (in green) and Hoechst 33342 for DNA labeling (in gray). Colocalization of both labeling (DNA/NE +) was represented in light green. (E) Graph depicts the percentage of NETs formation from human neutrophils in each condition (mean ^+/− SEM). PAF-activated neutrophils condition was used as positive control corresponding to 100% of NETs formation (ANOVA test, Dunnett multiple comparison, ****P < 0.0001). Three independent experiments with a minimum of five fields recorded for each condition.