Neutrophil extracellular traps were quantified by IHC staining of 2.5 × 104 neutrophils per well in a 96-well plate, mouse anti-human MPO antibody (1:750, ab25989, Abcam) and rabbit anti-human citH3 antibody (1:150, ab5103, Abcam), or the respective isotype controls, followed by incubation with goat anti-mouse IgG AF555 (1:1,000, A28180, Invitrogen Life Technologies) and goat anti-rabbit IgG AF488 (1:1,000, A11034, Invitrogen Life Technologies) (24, 38). DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich). NETs were visualized by using an Olympus IX81 motorized epifluorescence microscope (Olympus) in conjunction with an Olympus XM10 monochromatic CCD camera (Olympus) and analyzed with the Olympus CellSens Dimension software (Olympus). A minimum of 20 fields at 10× magnification (at least 500 to 1,000 neutrophils) per sample were evaluated for MPO/citH3 and DNA co-staining through ImageJ analysis software (NIH); nuclear phenotypes and NETs were determined, counted, and expressed as percentage of the total area of cells in the fields. For staining and quantification of NE, 1 × 105 neutrophils were seeded on poly-l-lysine-coated glass coverslips (BD Biosciences) in 24-well tissue-culture plates, fixed with 4% paraformaldehyde, and blocked overnight (HBSS with 10% FBS, 0.1% Tween20, and 2 mM EDTA) at 4°C. Samples were stained with rabbit anti-NE (1:200, ab26154, Abcam) following incubation with goat anti-rabbit IgG AF488 (1:1,000, Invitrogen Life Technologies). DNA was stained with DAPI (1:10,000, Sigma-Aldrich). Coverslips were visualized using an Olympus BX61 Diana fluorescent microscope.