Rapid Quantification of NETs In Vitro and in Whole Blood Samples by Imaging Flow Cytometry

Abstract

Neutrophil extracellular trap (NET) formation involves the release of DNA outside the cell to neutralize pathogens. Techniques such as live microscopy, flow cytometry, and intravital imaging allow the characterization of NETs, but these either cannot be applied in vivo, lack specificity or require invasive procedures. We developed an automated analysis method to rapidly acquire and characterize cells as NETs or NET precursors, as opposed to cells undergoing other forms of cell death, using imaging flow cytometry. NETs were maintained in solution using a novel three‐dimensional cell culture system in which cells are suspended at the interface of two liquids of different density. Critically, we identify NETs using an image analysis algorithm based on morphological data showing the extrusion of DNA beyond the cell boundaries. In vitro, we used this technique to demonstrate different requirements for NET formation in human and mouse neutrophils. We also measured NETs in whole blood during infection of mice with the malaria parasite Plasmodium yoelii. We expect this technique will provide a valuable approach to better understand the process of NET formation and its importance in disease. © 2019 International Society for Advancement of Cytometry

Distinguishing Sepsis From Infection by Neutrophil Dysfunction: A Promising Role of CXCR2 Surface Level

“…NETosis was identified by the presence of cell-appendant neutrophil extracellular traps components, including MPO and extracellular DNA. Extracellular DNA was detected by using the software masking features as previously described ( 19 ). These masking features identify cells with DNA contents located extracellularly and help exclude the cells stained by PI intracellularly (late apoptotic and necroptotic cells).…”

Quantitative Spectrofluorometric Assay Detecting Nuclear Condensation and Fragmentation in Intact Cells

“…Hoechst 33342 11,22 , 34580 23 or 33258 21 . Hoechst 33342 has been mostly used for ow cytometry [32][33][34] . On the other hand, H33258 staining has been predominantly used in uorescence microscopy 18,19,35 .…”

Monoclonal antibody 2C5 specifically targets neutrophil extracellular traps

“…Lelliott et al recently developed an imaging flow cytometry automated analysis method to characterize and quantify NETs in vitro based on DNA staining. 43 In their study, they characterized different cell types and NET structures after exposure to NET stimulants. Besides the extracellular DNA (exDNA) and DNA fragments characteristic of NETs, they noticed the presence of SYTOX-stained cells with decondensed DNA and an absence of exDNA.…”