Simplified 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. Surprisingly, there remains a paucity of 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. For the core assay, isolated primary human neutrophils were incubated with stimulants e.g. PMA/ionomycin with or without inhibitors prior to fixing. The fixed cells were then blocked and subsequently incubated in anti-DNA/Histone 1 and anti-histone H2A antibodies for dual detection of Histone 1 and Histone H2A. Cells with were H1-DNA/H2A double fluorescent were deemed NETotic. Imaging flow cytometry was used to validate the accuracy of NETosis detection/quantification. 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 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.