Fig. 2

Representative immunofluorescent images of cytology samples from 3 septic and 2 non-septic dogs. Cells were fixed, permeabilized and stained for citrullinated histone H3 (citH3, red), myeloperoxidase (MPO, green) and DNA (blue). NETs were identified by co-localization of decondensed cell-free DNA (cfDNA), citH3 and MPO. a, b Endotracheal wash from a dog with aspiration pneumonia. Note the vast amount of decondensed DNA decorated with extracellular citH3 and MPO surrounding nearby neutrophils (dotted outline). Activated pulmonary macrophages also were identified (*). c In the respective phase contrast image, phagocytized bacteria within activated pulmonary macrophages (*) and bacteria (arrow) within a NET (dash outline) were detected. NETs (dotted outline) were identified in septic abdominal (d) and pleural fluid (e). e, f The respective phase contrast image showed bacteria within a NET (dotted outline) and phagocytized bacteria within macrophages (*). Some neutrophils in septic effusion (a, e) had chromatin stained positive for citH3 (arrow heads). Non-degenerate neutrophils and activated macrophages were seen in abdominal fluid acquired from a dog with congestive heart failure (g) and a dog with chronic bronchitis (h); no NETs were identified. i A grayscale image stained for MPO and DNA demonstrating the measurement of cell diameter and nuclear morphology. Diameter of cell 1 was measured to be 8 μm. Cell 1 was identified to be a neutrophil based on its lobulated nuclei. Cell 2 was identified to be an activated pulmonary macrophage with a cell diameter of 18.5 μm, with rounded non-lobulated nuclei and presence of cytoplasmic vacuoles. Original 40× magnification. a–h Scale bar = 100 μm; (i) Scale bar = 50 μm