We thank the Light Imaging Section in the Office of Science and Technology at the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health. This research was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (ZIA AR041199).

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
<ol>
	<li>Take peripheral blood with appropriate written informed consent following the guideline of each institute and isolate neutrophils using any desired met...

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

<ol>
	<li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Wigerblad, G., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+systemic+autoimmune+and+autoinflammatory+diseases.">Neutrophil extracellular traps in systemic autoimmune and autoinflammatory diseases.</a> Nat Rev Immunol. 23 (5), 274-288 (2022).</li><li>Wagner, D. D., Heger, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thromboinflammation:+From+atherosclerosis+to+COVID-19.">Thromboinflammation: From atherosclerosis to COVID-19.</a> Arterioscler Thromb Vasc Biol. 42 (9), 1103-1112 (2022).</li><li>Njeim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NETosis+contributes+to+the+pathogenesis+of+diabetes+and+its+complications.">NETosis contributes to the pathogenesis of diabetes and its complications.</a> J Mol Endocrinol. 65 (4), R65-R76 (2020).</li><li>De Meo, M. L., Spicer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neutrophil+extracellular+traps+in+cancer+progression+and+metastasis.">The role of neutrophil extracellular traps in cancer progression and metastasis.</a> Semin Immunol. 57, 101595 (2021).</li><li>Nakabo, S., Romo-Tena, J., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+as+drivers+of+immune+dysregulation+in+autoimmune+diseases+with+skin+manifestations.">Neutrophils as drivers of immune dysregulation in autoimmune diseases with skin manifestations.</a> J Invest Dermatol. 142 (3 Pt B), 823-833 (2022).</li><li>Gupta, S., Chan, D. W., Zaal, K. J., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+real-time+imaging+technique+to+quantify+NETosis+and+distinguish+mechanisms+of+cell+death+in+human+neutrophils.">A high-throughput real-time imaging technique to quantify NETosis and distinguish mechanisms of cell death in human neutrophils.</a> J Immunol. 200 (2), 869-879 (2018).</li><li>Nakabo, S., Kaplan, M. J., Gupta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+neutrophils+undergoing+NET+formation+and+distinguishing+mechanisms+of+neutrophil+cell+death+by+use+of+a+high-throughput+method.">Quantification of neutrophils undergoing NET formation and distinguishing mechanisms of neutrophil cell death by use of a high-throughput method.</a> Methods Mol Biol. 2543, 129-140 (2022).</li><li>Singh, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moonlighting+chromatin:+when+DNA+escapes+nuclear+control.">Moonlighting chromatin: when DNA escapes nuclear control.</a> Cell Death Differ. 30 (4), 861-875 (2023).</li><li>Carmona-Rivera, C., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+quantification+of+NETosis.">Induction and quantification of NETosis.</a> Curr Protoc Immunol. 115, 14.41.11-14.41.14 (2016).</li><li>Hsu, A. Y., Peng, Z., Luo, H., Loison, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+neutrophils+from+whole+blood+and+buffy+coats.">Isolation of human neutrophils from whole blood and buffy coats.</a> J Vis Exp. (175), 62837 (2021).</li><li>Neubert, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+and+serum+albumin+inhibit+in+vitro+formation+of+neutrophil+extracellular+traps+(NETs).">Serum and serum albumin inhibit in vitro formation of neutrophil extracellular traps (NETs).</a> Front Immunol. 10, 12 (2019).</li><li>von Kockritz-Blickwede, M., Chow, O. A., Nizet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+calf+serum+contains+heat-stable+nucleases+that+degrade+neutrophil+extracellular+traps.">Fetal calf serum contains heat-stable nucleases that degrade neutrophil extracellular traps.</a> Blood. 114 (25), 5245-5246 (2009).</li><li>Zhao, W., Fogg, D. K., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+image-based+quantitative+method+for+the+characterization+of+NETosis.">A novel image-based quantitative method for the characterization of NETosis.</a> J Immunol Methods. 423, 104-110 (2015).</li><li>Papayannopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+immunity+and+disease.">Neutrophil extracellular traps in immunity and disease.</a> Nat Rev Immunol. 18 (2), 134-147 (2018).</li></ol>

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 way10,14. 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&#39;s subjective view. A plate assay detecting the fluorescence of membrane-imp...

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 platform7,8. 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 PKC&#945;-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AKT inhibitor</td>
			<td>Calbiochem</td>
			<td>124028</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear 96-well plate</td>
			<td>Corning</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr>
			<td>Live cell analysis system</td>
			<td>Sartorius</td>
			<td>N/A</td>
			<td>Incucyte Software (v2019B)</td>
		</tr>
		<tr>
			<td>Membrane-impermeable DNA green dye&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear red dye</td>
			<td>Enzo</td>
			<td>ENZ-52406</td>
			<td>Neutrophil pellet becomes bluish after staining.</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Thermo Fisher Scientific</td>
			<td>11835030</td>
			<td>Phenol red containig RPMI can be used.</td>
		</tr>
	</tbody>
</table>

real-time high throughput technique to quantify neutrophil extracellular traps formation in human neutrophils

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.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

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

Our research focuses on neutrophils, which are essential first responders in the immune system and play a significant role in various diseases. Over the past two decades, it has become clear that neutrophils contribute to the development of cancer, autoimmune diseases, and other inflammatory conditions by disrupting immunoregulation. This includes forming neutrophilic extracellular traps, nets, web-like structures that respond to inflammation and could be targeted for therapy in these diseases.However, despite some promising molecules targeting nets, in development there is still no approved therapy that specifically affects this mechanism. This is at least partially attributable to the lack of an objective, unbiased, reproducible, and high-throughput quantification method for net formation. Our protocol employs dual-color live cell imaging to analyze neutrophil behavior using membrane permeable and impermeable dyes for precise net formation tracking.This method distinguishes between net-forming and healthy neutrophils based on membrane integrity and provides clear differentiation of cell death types through morphological changes absorbed in phase contrast imaging. This method overcomes the problems of previously-reported techniques to quantify net formation and provides an efficient, reproducible, and accurate net quantification in an automated manner. This method will help in neutrophil-targeted drug development by giving the ability to screen multiple therapeutic targets against net formation in a high-throughput manner.

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...

Watch this Scientific Journal Video about Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging at JoVE.com

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.