Measurement of NET formation in vitro and in vivo by flow cytometry

Abstract

Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane‐impermeable DNA‐binding dye, SYTOX Green. SYTOX Green‐positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12‐myristate 13‐acetate (PMA). The number of SYTOX Green‐positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co‐localization of MPO and plasma membrane‐appendant DNA of SYTOX Green‐positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green‐positive cells induced by PMA. The collective evidence suggests that SYTOX Green‐positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well‐used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

A Flow Cytometry‐Based Assay for High‐Throughput Detection and Quantification of Neutrophil Extracellular Traps in Mixed Cell Populations

“…A side by side comparison with the assay by Gavillet et al revealed greater sensitivity in the detection of NETosis with our protocol (Fig. 3B and Supporting Information (21,25). Similar to our findings, percentage of SG + cells correlated with NET area stained using DAPI and observed on fluorescent microscopy (25).…”

“…However, the assay by Nel et al can underestimate the earlier phases of NETosis when the NETs are still PMN‐appendant. Additionally, Masuda et al utilized Sytox Green (SG), a plasma membrane‐impermeable DNA dye, to detect PMN‐appendant NET‐DNA . Similar to our findings, percentage of SG + cells correlated with NET area stained using DAPI and observed on fluorescent microscopy .…”

“…3B and Supporting Information (21,25). Similar to our findings, percentage of SG + cells correlated with NET area stained using DAPI and observed on fluorescent microscopy (25). However, SG + cells were gated solely based on FSC and SSC properties, which limit the assay to purified populations and may include debris (54).…”

“…In this report, we describe an optimized protocol utilizing a combination of SO and 4 0 ,6-diamidino-2-phenylindole (DAPI) for two-color visualization of NETs on flow cytometry (20). SO + DAPI + PMNs activated with phorbol 12-myristate 13-acetate (PMA) was dose-and time-dependent, as previously described (8,25). To verify that SO + DAPI + PMNs were not due to other forms of cell death, we determined the following findings: (1) minimal SO + DAPI + PMNs with epoptoside-induced apoptosis; (2) correlation in the percentage of SO + DAPI + PMNs with NETosis rate obtained using time-lapse live cell microscopy; and (3) colocalization of extracellular DNA and NE on flow cytometry.…”

Quantification of NETs‐associated markers by flow cytometry and serum assays in patients with thrombosis and sepsis

“…Flow cytometric assays for direct quantification of cell‐bound NETs on isolated neutrophils has recently been described in a limited number of patients . The purpose of this study was to develop a method of NETs marker quantification for potential routine application using whole blood flow cytometry.…”