Establishment of a multi-parameter flow cytometry method to identify and characterize neutrophil extracellular traps

Abstract

Neutrophil extracellular traps (NETs) are web-like decondensed DNA filaments released by activated neutrophils, decorated with antimicrobial proteins such as myeloperoxidase (MPO) and elastase. Although several methods exist to evaluate NETs formation, including fluorescent microscopy or scanning electron microscopy (SEM), and flow cytometry, each has inherent limitations that restrict widespread application. Given the increasing relevance of NETs in various pathophysiological contexts, we sought to develop a simple, specific, objective and cost-effective flow cytometry-based method to assess NETs both in vitro and in vivo. Our approach leverages multi-parametric flow cytometry to simultaneously evaluate cell size, granularity, DNA decondensation, histone citrullination, and intracellular MPO. This method enables reliable detection of NETs in purified neutrophils as well as in tissue samples. Its performance was validated in parallel with conventional microscopy, confirming specificity and reproducibility. Notably, this FACS-based method is faster, more economical, and free from observer-bias, making it especially well-suited for both research and clinical sample analysis.