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 method. For example, the Ficoll-dextran method10,11 is a commonly used method for isolation of neutrophils from human peripheral blood. 
	NOTE: Although the method discussed here uses human neutrophils, mouse neutrophils may also be analyzed using a similar protocol.</li>
	<li>Resuspend neutrophils in Roswell Park Memorial Institute (RPMI; see Table of Materials) 1640 medium at 2.0 x 106 neutrophils/mL and place neutrophil suspension into 1.5 mL centrifuge tube. 
	NOTE: We usually do not add fetal bovine serum (FBS) to the culture media because albumin in FBS can reduce NET formation12 and heat-stable nuclease in FBS can degrade NETs13.</li>
	<li>Add membrane-permeable red DNA dye (see Table of Materials) at 1 &#181;L per 1.5 mL of neutrophil suspension.</li>
	<li>Incubate at room temperature for 5 min in the dark. Centrifuge at 2500 x g for 5 min at room temperature and remove supernatant.</li>
	<li>Resuspend neutrophils in 1 mL of RPMI, then centrifuge at 2500 x g for 5 min at room temperature and remove supernatant. Repeat 2x (wash total 3x).</li>
	<li>Resuspend neutrophils in 1 mL of RPMI and count using a cell counter. Dilute neutrophil suspension to 1.5 x 105 neutrophils/mL.</li>
	<li>Add 4 &#181;L of 1:100-pre-diluted membrane-impermeable green DNA dye (see Table of Materials) per 1 mL of neutrophil suspension.</li>
	<li>Put 100 &#181;L of neutrophil suspension per well into a clear tissue-culture treated 96-well plate (see Table of Materials). Set each condition in triplicate. 
	NOTE: We have previously shown7 that there is no difference in NET formation and quantification with this method if neutrophils are placed in plates with or without poly-L-lysine coating. Therefore, the use of tissue culture treated plate is sufficient for this method.</li>
	<li>Add 100 &#181;L of RPMI containing stimulus reagents with or without inhibitor, or any other reagent of interest in respective wells. Always use positive control wells by adding 500 nM phorbol 12-myristate 13-acetate (PMA) or 2.5 &#181;M calcium ionophore (A23187), 30 &#181;M AKT inhibitor was used here.</li>
	<li>Place plate in the live cell analysis system (see Table of Materials) that is housed in a 5% CO2 incubator. 
	NOTE: Condensation may appear on the top and bottom of the plate a few minutes after this step. This may inhibit proper imaging. Wipe and remove condensation thoroughly before the start of the scanning. Place the plate in the machine, start the software, input scanning protocol, and then wipe the plate just before the first scan.</li>
</ol>
2. Scanning plate to visualize NET-forming neutrophils
<ol>
	<li>Start the software (see Table of Materials for software details). Launch Add Vessel by pressing + at the top left corner.</li>
	<li>Choose Scan on Schedule. Choose New. 
	NOTE: Once this is created, run the same scanning protocol by choosing Copy Previous and selecting stored protocol on the next screen.</li>
	<li>Choose Standard. Set scan settings as: Cell-by-Cell options: None; Image channels: Phase, Green (Acquisition Time: 200 ms), Red (Acquisition Time: 400 ms); Objective: 20x.</li>
	<li>Choose the plate being used from the list. Select vessel location where to put the plate on the tray of the live cell imaging system.</li>
	<li>Select wells where samples are present and decide how many images to take per well. Based on the number of images per well, generate an estimated scan duration for the plate. Usually, 4 images per well is sufficient; however, this may vary depending on the conditions and frequency of scans.</li>
	<li>Input the information from each well (e.g., cell type and compound) by clicking Create Plate Map to provide a name for the study. 
	NOTE: The plate map can be prepared and saved in advance using the plate map editor in the software. The saved plate map data can be imported during this step. If desired, entering plate map information can be skipped and performed later. Data can also be obtained without entering information of plate layout. However, it is recommended to input plate information to make the subsequent analysis easier.</li>
	<li>On the next screen, select Basic Analyzer as the analysis type, and choose Analysis Protocol from drop-down list, which shows previously used analysis definitions (not scanning protocols). Spectral unmixing for both green and red can be left at 0.0 %. 
	NOTE: This step may be skipped, if desired.</li>
	<li>Schedule scan. For the experiment here, scan at every 15-20 min for 8 h . Set the starting time of the scan by dragging the white and gray bar on top of the screen. 
	NOTE: Avoid scanning too frequently, otherwise the machine may not work well due to overheating. Time of scanning should not exceed 12 h per 24 h. You may see an alert if scanning is too frequent.</li>
	<li>Check the scan setting on the next screen and press Add to Schedule to start scanning. Wait until the initial set of images are scanned to confirm if everything is working well.
	<ol>
		<li>Sometimes cells may not be properly focused. In that case, check if condensation is present (and wipe accordingly), plate is correctly set on the tray, or the position of the plate is correctly specified, etc. If cells are still floating and have not settled down to the bottom of the well, wait for about 5 min before scanning.</li>
	</ol>
	</li>
</ol>
3. Setting the analysis definition to quantify NETs
<ol>
	<li>Open the study (vessel) to be analyzed on View tab. Press Launch Analysis on the left of the screen. Choose Create New Analysis Definition.
	<ol>
		<li>If an analysis has been performed previously, use the same analysis definition with minor alterations by selecting Copy Existing Analysis Definition. In this case, go to step 3.3. If using the analysis without any alteration select Use Existing Analysis Definition; this is not recommended because the level of fluorescence may vary between assays on different days, and some minor corrections are usually necessary.</li>
	</ol>
	</li>
	<li>Select Basic Analyzer. Use all image channels: Phase, Green, Red, and Overlap. 
	NOTE: Although we usually use green and red object masks to count NETs, overlap object mask is useful when debris matters. In such cases, the number of overlap object count will be used as numerators instead of green object count (see step 3.9). If overlap object count is used instead of green object count, all red signals must be counted correctly. Adjust the setting of red signal to count all red objects with low red signal in images taken in later timepoints but not to count debris.</li>
	<li>Choose 6 - 8 representative sample images for training. For the experiment here, include the following images: 
	Images taken between 0 to 20 min to optimize the green signal setting to compensate for subtle green signals that can be generated in non-NET-forming neutrophils 
	Images of maximum NETs with positive control, taken between 3-6 h (time varies depending on the stimulation used) to optimize the green signal setting to define NET-forming neutrophils 
	Images taken around the 1 h timepoint to count the number of total cells (stained with red dye) since the nuclear red signal is the strongest around 1 h timepoint (when all the cells have settled to the bottom of the well) and gradually decreases due to cell death. 
	Images containing debris, to exclude them from being counted.</li>
	<li>Set the analysis definition. Red objects and green objects correspond with nuclei of all neutrophils and those that are forming NETs, respectively. Start with the following example of and press Preview Current or Preview All, and modulate each parameter to optimize results: 
	For Green: Segmentation- Background subtraction: Top-Hat; Radius: 100 &#181;M; Threshold: 0.3 GCU; Edge split: On; Edge sensitivity: -20; Cleanup -Hole fill: 100 &#181;m2; Adjust size 0 pixels; Filters- Area: min 20 &#181;m2, max 500 &#181;m2; Eccentricity: max 0.97; Mean Intensity: min 1.00 
	&#8203;For Red: Segmentation- Background subtraction: Top-Hat; Radius: 10 &#181;M; Threshold: 1.0 GCU; Edge split: ON; Edge sensitivity: -50; Cleanup-Hole fill: 50 &#181;m2; Adjust size 0 pixels; Filters- Area: min 20 &#181;m2, max 400 &#181;m2; Mean Intensity: min 1.5.
	<ol>
		<li>If excessive green signal appears in neutrophils which should not be forming NETs (e.g., unstimulated neutrophils at 0 min that have lobulated nuclei), it may be due to overflow of the red signal detected in the green channel. In such case, return to step 3.1, open Image Layers on the left of the screen, and remove the red signals from the green channel by using Spectral Unmixing.</li>
		<li>Another option is to set one well for single staining with nuclear red dye to clarify how much of the red signal should be removed from green channel. On the other hand, usually green signal bleeding into the red channel does not affect the analyses. 
		NOTE: It does not matter if the sensitivity to red signal is low and not all red signals are captured in the images that are taken at later timepoints (e.g., 6 h timepoint). The maximum number of red object count in each image is regarded as total number of neutrophils in the image and will be used as denominator at step 3.9. Usually, nuclear red signals peak around 1 h timepoint and gradually diminish due to cell death (Figure 1B). Therefore, adjust the setting of red signal to correctly count red objects in the images with maximum red signals.</li>
	</ol>
	</li>
	<li>Select scan times and wells to be analyzed. Usually, all time points and wells should be analyzed. Provide a label to the analysis definition.</li>
	<li>Check the summary and launch the analysis. It takes a few hours for completion of analysis (the duration depends on the number of timepoints and wells). Once launched, the name of the analysis definition will be shown under the name of the study in the View tab, and the complete date will be shown when it is done.</li>
	<li>After it is completed, open the analyzed study by double clicking the name of the analysis definition. Open Layers on the left of the screen and check if each cell is correctly marked. If it is not correctly marked, return to step 3.1 and repeat the subsequent steps.</li>
	<li>Click Graph Metrics on the left of the screen to export the data. Select Green Count, Red Count or Overlap Count (Per Image), and then choose the timepoints and wells to be exported. For select grouping, select Plate Map Replicates if all wells are correctly specified when scanning is done.</li>
	<li>Export the data. Calculate the percentage of NET-forming cells for each condition/timepoint by the following equation using an appropriate software. 
	<img alt="Equation 1" src="/files/ftp_upload/66051/66051eq01.jpg" style="margin: auto;" /> 
	NOTE: The reason why the denominator is maximum red object count is that the number of Red object peaks at around 1 h timepoint and gradually decreases due to the cell death.</li>
</ol>

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-impermeable DNA dye quantifies extracellular DNA as a surrogate marker of NETs in an objective and high-throughput manner, but since morphological information cannot be obtained by this method, DNA release by other types of cell death may be wrongly regarded as NETs. On the other hand, the method provides high-throughput and objective quantification of NETs7. Given that information about morphological changes of neutrophils and its time course can be obtained, NETs are accurately discriminated from other types of cell death. Recent papers have suggested pathogenic role of NETs in various diseases2,3,4,5, and inhibition of NETs is currently regarded as a potentially promising treatment target6. The method will be beneficial to explore multiple NET-targeting molecules in a rapid and impartial way.
There are several key points for successful quantification. Choice of membrane-permeable DNA binding dye is a very important factor for proper imaging. Some dyes are cytotoxic, and other dyes take time to permeate into nuclei. Since NET formation is relatively rapid, the dye must permeate into the nuclei quickly. We chose a nuclear red dye (see Table of Materials) taking these factors into consideration. The number of cells in each well is also important for accurate counting. If it is too crowded, green and red signals of each neutrophil will overlap with each other, which makes it difficult to separately count each neutrophil.
There are a few limitations to this method. While intra-assay variability is excellent in this assay, inter-assay variability is unclear. This is because neutrophils, even when isolated from the same donor with all conditions kept consistent, act differently on different days and there may be some variation between assays performed on another day. Therefore, if data from different days needs to be combined, it is necessary to carefully design the assay: for example, to include the same number of samples from disease and control groups on each day. In addition, although the evaluation of NET formation is objective once the analysis definition is defined (step 3), setting the definition itself somewhat depends on the subjective view of each operator. Steps 3.3 and 3.4 are the critical steps to exclude subjective judgments and keep inter-assay variability as small as possible. Also, a positive control (e.g., neutrophils stimulated by 500 nM PMA or 2.5 &#181;M calcium ionophore) must be included every time, to set the appropriate cut-off to count all NETs.
On the other hand, it must be noted that other types of cell death may be counted as NETs. When the cell membrane is breached, or DNA is released to extracellular space, green signals will be observed. When cells undergo necrotic cell death or apoptotic cells undergo secondary necrosis, their DNA will be stained by green dye7. To prevent inaccurately including these types of cell death, time course and morphological changes must be considered. While it usually takes more than 8 h for apoptotic cells to perform secondary necrosis, NET formation peaks between 3-6 h after stimulation15. Distinct morphological changes can be seen by phase contrast imaging, which is beneficial for differentiation of type of cell death7.
Overall, the method enables us to accurately quantify NETs in a high throughput and objective manner.

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.

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 machine is not high enough to capture fine web-like structure and membrane impermeable green dye stains chromatin before it is released once the membrane is breached. We have previously shown7 that NETs can be visualized by use of confocal imaging in the 96-well plates retrieved after 4 h incubation.
When the analysis definition is appropriately set, all neutrophils in the image are marked as red object and NET-forming neutrophils are marked as green object (Figure 2). The machine counts the number of red and green objects at each time point. The time course of NET formation is visualized by plotting the percentage of NET-forming neutrophils at each time point (Figure 3). Potential molecules that target NET formation (e.g., AKT inhibitor) may be tested in a high throughput manner using this methodology.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66051/66051fig01.jpg" /> 
Figure 1: Morphological changes in neutrophils undergoing NET formation. (A) Human peripheral blood neutrophils that were stimulated with 2.5 &#181;M calcium ionophore for 3 h. (B) Representative single-cell views of phase contrast image, red channel (membrane-permeable nuclear dye), green channel (membrane-impermeable DNA dye), and merged image. <a href="https://www.jove.com/files/ftp_upload/66051/66051fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66051/66051fig02.jpg" /> 
Figure 2: Representative images showing software recognition of neutrophils and NETs. Neutrophils from human healthy volunteers were stimulated with 2.5 &#181;M calcium ionophore for 1 h. Overlaid images of phase contrast imaging and each signal or mask are shown. Nuclei were stained with (A) membrane-permeable red dye, and the software recognized and counted (B) nuclei marked blue while the NETs were stained with (C) membrane-impermeable green dye and the software marked them as (D) purple. If (E) oversensing or (F) under sensing occurs, parameter may need to be changed. <a href="https://www.jove.com/files/ftp_upload/66051/66051fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66051/66051fig03.jpg" /> 
Figure 3: Time course of the percentage of NET-forming neutrophils. Neutrophils were stimulated by 25 nM phorbol 12-myristate 13-acetate (PMA) or 2.5 &#181;M calcium ionophore to induce NETs or left unstimulated in RPMI. Addition of 30 &#181;M AKT inhibitor was done to block NET formation. Images were obtained by the software every 20 min for 6 h. The percentage of NET-forming cells was calculated by dividing the green object count (= the number of NET-forming cells) by the red object count (= the number of all neutrophils). <a href="https://www.jove.com/files/ftp_upload/66051/66051fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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.

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 ...

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862 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.

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...