Detection, visualization and quantification of Neutrophil Extracellular Traps (NETs) and NET markers
Neutrophil extracellular traps (NETs) have been identified as a key player in the pathogenesis of infection and inflammation in human and animals. On the one hand, NETs have been characterized as fundamental to the innate immune defense against different pathogens since they are able to entrap and immobilize invading pathogens. On the other hand, NETs have been shown to contribute to several diseases, based on their detrimental consequences. This chapter describes methods to detect NETs and NET markers in blood-derived isolated neutrophils of human, pigs, and horses in vitro, as well as NETs and NET marker detection in body fluids from in vivo studies. To avoid nonspecific background in NET-formation, a well-established isolation method for the neutrophils from fresh blood is needed. After stimulation of neutrophils to release NETs, NETs are stained with different antibodies to confirm the presence of extracellular DNA extrusion consisting of histone-DNA complexes, as well as granule components (e.g., myeloperoxidase or elastase). Furthermore, specific methods to quantify NETs and NET markers in the cerebrospinal fluid (CSF) and bronchoalveolar lavage fluid (BALF) are described in detail. In addition to immunofluorescence microscopy, quantification of NET markers from in vivo experiments in various body fluids is described (e.g., nuclease activity, free extracellular DNA, or cationic host defense peptides, such as the porcine PR-39 in BALF and CSF).