Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Summary

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Abstract

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Introduction

Neutrophil extracellular traps (NETs) are web-like chromatin structures extruded from neutrophils in response to various inflammatory stimuli. NETs are composed of DNA, histones, and various anti-microbial proteins/peptides, which trap and kill infectious pathogens and invoke inflammatory responses¹.

While NETs are beneficial for host defense against pathogens, they have gathered attention as a potential driver of various autoimmune diseases², thrombosis³, metabolic diseases⁴, and metastatic growth of cancers⁵. As such, inhibition of NET formation is a potential therapeutic option for these diseases. However, despite some promising NETs-targeting molecules in development⁶, there is still no approved therapy that specifically affects this mechanism. This is, at least partially, attributable to the lack of objective, unbiased, reproducible, and high throughput quantification methods for NET formation.

We established and reported a new method utilizing a dual-color live-cell imaging platform^7,8. Time-lapse images of neutrophils stained with membrane-permeable nuclear dye and membrane-impermeable DNA dye are analyzed by the software, and the numbers of pre- and post-NET-forming neutrophils are counted at multiple time points. Since the integrity of the plasma membrane is lost during NET formation by the regulation of PKCα-mediated Lamin B and CDK4/6-mediated Lamin A/C disassembly⁹, NET-forming neutrophils are stained by membrane-impermeable DNA dye while healthy neutrophils are not. This method overcomes the problems of previously reported techniques to quantify NET formation and provides unbiased, high-throughput, reproducible, and accurate NET quantification in an automated manner.

Protocol

Neutrophils from healthy human subjects were obtained after informed consent was provided under National Institutes of Health (NIH) Institutional Review Board (IRB) approved protocol. The protocol follows guidelines of NIH human research ethics committee. 1. Staining of the neutrophils and preparation of assay plate Take peripheral blood with appropriate written informed consent following the guideline of each institute and isolate neutrophils using any desired met…

Representative Results

This method provides phase contrast, red fluorescent (membrane-permeable dye) and green fluorescent (membrane-impermeable dye) images taken at each timepoint. Along with the NET-forming process, morphological changes are observed in phase contrast and red fluorescent images, and once the membrane is breached, green fluorescence can be observed (Figure 1). In this assay, NET-forming neutrophils are generally round, instead of forming web-like structure. This is because the resolution of the m…

Discussion

Current methods to quantify NETs ex vivo have several drawbacks that limit our ability to study neutrophils, NETs, and potential therapeutic targets in an unbiased and high-throughput way^10,14. For example, direct counting of NET-forming cells after immunofluorescent staining, considered the gold standard for quantification of NETs, is low-throughput and dependent on operator's subjective view. A plate assay detecting the fluorescence of membrane-imp…

Materials

AKT inhibitor	Calbiochem	124028
Clear 96-well plate	Corning	3596
Live cell analysis system	Sartorius	N/A	Incucyte Software (v2019B)
Membrane-impermeable DNA green dye	Thermo Fisher Scientific	S7020
Nuclear red dye	Enzo	ENZ-52406	Neutrophil pellet becomes bluish after staining.
RPMI	Thermo Fisher Scientific	11835030	Phenol red containig RPMI can be used.