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>

Authors have no competing financial interests.

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.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

real-time-high-throughput-technique-to-quantify-neutrophil

Research

JoVE Journal

Immunology and Infection

Human Neutrophil Staining and Preparation of Neutrophil Assay Plate for Live Cell Analysis

After isolating neutrophils from human peripheral blood, resuspend them in RPMI 1640 Medium in a 1.5 milliliter centrifuge tube. Add one microliter of membrane permeable red DNA die to 1.5 milliliters of neutrophil suspension and incubate in the dark for five minutes. Centrifuge the neutrophil suspension at 2, 500g for five minutes at room temperature.After centrifugation, remove the tube containing pelleted neutrophils and aspirate the supernatant. Resuspend the neutrophils in one milliliter of RPMI and centrifuge it again. After the last wash, resuspend the neutrophils in one milliliter of RPMI.Add a small volume of neutrophil suspension to the hemocytometer and count them under a microscope. Dilute the neutrophil suspension to 1.5 times 10 to the fifth neutrophils per milliliter using RPMI. Add four microliters of one to 100 pre-diluted membrane impermeable green DNA dye to the one milliliter of neutrophil suspension.Dispense 100 microliters of neutrophil suspension per well into a clear tissue culture treated 96 well plate. Add 100 microliters of RPMI containing stimulus reagents with or without inhibitor into the respective wells. Place the plate in a live cell analysis system housed in a 5%carbon dioxide incubator.

Scanning Plate Setup and Quantitative Analysis of Neutrophil Extracellular Traps Formation in Human Neutrophils

To begin, start the live cell analysis system software. Press the plus symbol at the top left corner of the screen to initiate the add vessel. Choose Scan on Schedule, then select New to run the scanning program.Select Standard. Then set scan settings as cell-by-cell options to None and image channels to Phase, Green, and Red, with respective acquisition times. Set objective to 20x.From the list, choose the plate and select the vessel location to put the plate on the tray of the imaging system. Select the wells containing samples and decide the number of images to capture per well. This information automatically generates an estimated scan duration for the plate.Click Create Plate Map to input information for each well, such as cell type and compound. Then name the study. On the next screen, select Basic Analyzer as the analysis type and choose Analysis Definition from the dropdown list, which shows previously used analysis definitions.Leave spectral unmixing for both green and red at 0.0. Schedule the scan to occur every 15 to 20 minutes for eight hours. Drag the white and gray bar on top of the screen to set the starting time of the scan.On the next screen, review the scan settings, and press Add to Schedule to start scanning. After scanning, on the View tab, open the vessel for analysis. Press Launch Analysis on the left of the screen and select Create New Analysis Definition.Choose Basic Analyzer and use Phase, Green, Red, and Overlap image channels. Select six to eight representative sample images for training. To configure the analysis definition, set green objects to neutrophils forming neutrophil extracellular traps or NETs, and red objects to the nuclei of all neutrophils.Press Preview Current or Preview All and adjust each parameter to optimize the results. Choose the scan times and wells for analysis. Then provide a label for the analysis definition.Review the summary and launch the analysis. After analysis, double click on the name of the analysis definition to open the analyzed study. Open Layers on the left of the screen and check if each cell is correctly marked.Click Graph Metrics on the left of the screen, then select count per image for green count. Choose the time points and wells to be exported. For select grouping, select Plate Map Replicates to confirm all wells are correctly specified during scanning, and click on Export Data.Similarly, export the data for red count and overlap count. Using the given equation, calculate the percentage of NETs forming cells for each condition and time point. The time course of the NET formation is visualized by plotting the percentage of NET-forming neutrophils at each time point.Adding an AKT inhibitor blocked the NET formation in neutrophils stimulated with PMA or calcium ionophore. Demonstrating the potential molecules targeting NET formation may be tested in a high throughput manner using this methodology.

650 Views.  National Institutes of Health (NIH).  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.

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

Summary