A sensitive, fast, and efficient method for studying NETosis ex vivo by flow cytometry and live cell imaging 2450

Abstract Description In a process termed NETosis, neutrophils expel neutrophil extracellular traps (NETs) - chromatin fibers decorated with antimicrobials and proteases, including neutrophil elastase - in an effort to trap and eliminate pathogens. NETosis plays a role in the pathogenesis of multiple diseases, and standardized methods for analyzing and measuring the process are urgently needed for the advancement of this research area. Here we demonstrate a sensitive and streamlined method for the quantification of NETosis ex vivo by flow cytometry and live cell imaging. A neutrophil elastase-activated fluorescent probe is combined with membrane-permeable and impermeable DNA-binding dyes, allowing for the detection of the different stages of NETosis, such as nuclear swelling, the release of neutrophil elastase and extracellular DNA, and loss of intact nuclei. Using this method, we were able to detect a time-dependent increase in the number of neutrophils undergoing NETosis upon stimulation, while NETosis was not detected in untreated neutrophils. Our rapid, simple protocol reduces hands-on time and maximizes reproducibility. This assay is compatible with flow cytometry, facilitating high-throughput detection and quantification of NETosis in purified or mixed cell populations. This assay provides detailed morphological information and visualization of the distinct stages of NETosis while enabling discrimination between live cells, cells that have undergone NETosis, and dead or apoptotic cells. Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)