Automated flow cytometric quantification of human neutrophil extracellular traps (NETs)

Several cellular pathways lead to the formation of neutrophil extracellular traps (NETs), a form of cell death (NETosis), distinct from apoptotic and necrotic cell death. NETosis involves the controlled extrusion of extracellular chromatin laced with antimicrobial proteins, which collectively ensnare and kill microorganisms. However, dysregulation of NETosis can contribute to the pathogenesis of multiple diseases. Hence, NETosis is regarded as a ‘double-edged sword’ in immunity and thus has received significant research attention. Surprisingly, however, there remains a paucity of automated methods enabling efficient quantification of NETosis and associated pathways. Here, we describe the development of a simple, sensitive, reliable and flexible flow cytometry assay allowing efficient detection and quantification of NETosis. Firstly, imaging flow cytometry was used to validate the accuracy of NETosis detection/quantification by conventional antibody-based flow cytometry assay. Several key pathways of NETosis were confirmed via the use of established NET-inducers in accordance with existing established methodologies. Importantly, our novel flow cytometry-based NETosis detection assay could efficiently discriminate NETosis from established neutrophil activation, as well as apoptotic and necrotic cell death. We believe our automated methodology will complement existing NETosis methodologies whilst concomitantly reducing human error, subjectivity and, indeed the false positivity (attained from neutrophil activation and other cell death processes) inherent in existing methodologies. Furthermore, the simplicity and flexibility of our methodology permit additional markers and pathways of NETosis to be investigated, highlighting it as an integral research tool for both general NETosis research and the pursuit to better understand the pathogenesis of NETosis-associated diseases.