Neutrophils from PAD4^−/− mice are competent in non-NET functions. (A) Diluted, anticoagulated whole blood was incubated with 1 μM ionomycin, 100 nM PMA, or vehicle for 20 minutes at 37°C in the presence of dihydrorhodamine-123. Rhodamine⁺ ROS-generating neutrophils are quantified for each condition (left panel) and representative plots are shown (right two panels); n = 4-13. (B) Diluted whole blood was preincubated with 5 mM cytochalasin B for 20 minutes, then incubated with 1 μM ionomycin, 100 nM PMA, or vehicle for 10 minutes at 37°C. Lamp1⁺ Ly6G⁺ degranulated cells are quantified in the left panel, with representative plots shown in the right panels; n = 4-13. (C) Leukocyte recruitment was assessed in vivo using thioglycollate-induced peritonitis. Infiltrating cells were counted in peritoneal lavage fluid at the indicated time points (left panel) and differential counts performed at 4 hours from Wright Giemsa-stained cytospins to assess neutrophil infiltration (center panel). Representative 4-hour cytospin images are shown in the right panels.