The work of Eline J. Arends and Y.K. Onno Teng is supported by the Dutch Kidney Foundation (17OKG04), Clinical Fellowship from the Netherlands Organization for Scientific Research (90713460). Laura S. van Dam&#39;s work is supported by the Foundation for Research in Rheumatology (FOREUM).

All patients and healthy controls consented to participate in the LUMC biobank. Both biobanking studies were approved by the LUMC ethical committee.
1. Isolation of Healthy Neutrophils
<ol>
	<li>Obtain 20 mL of peripheral blood from a healthy donor in two 10 mL EDTA-coated tubes.</li>
	<li>Put 10 mL of blood in a sterile 50 mL tube and add phosphate buffered saline (PBS) up to 32.5 mL.</li>
	<li>Add density gradient (e.g., Ficoll-amidotrizoaat) under the cells.
	<ol>
		<li>Take up 14 mL of density gradient with a 10 mL pipet and pipet controller.</li>
		<li>Place the pipet on the bottom of the 50 mL tube.</li>
		<li>Take the pipet controller off the pipet, allowing the density gradient to flow out of the pipet by gravity until the maximum is reached by capillary effect (1-2 mL will be left), without using the motor.</li>
		<li>Remove pipet by placing a thumb on top of the pipet, thereby preventing density gradient from leaking out during removal of the pipet.</li>
	</ol>
	</li>
	<li>Spin the tubes for 20 min at 912 x g and room temperature (RT) without acceleration or brake. 
	NOTE: Red blood cells (RBC) and neutrophils have a high density and are at the bottom of the 50 mL tube. Peripheral blood mononuclear cells (PBMCs) are separated and on top of the density gradient as a white ring. PBS-diluted plasma will be above the PBMCs. If needed, PBMCs can be isolated by transferring the white ring to a new 50 mL tube with additional washing steps with PBS.</li>
	<li>Carefully remove the white ring containing PBMCs first, followed by removal of the PBS-diluted plasma and lastly the density gradient layer as much as possible.</li>
	<li>To isolate neutrophils from the neutrophil/erythrocytes mix, lyse erythrocytes by cold sterile distilled water.
	<ol>
		<li>Take cold sterile distilled water bottle and a 10x concentrated PBS flask from the fridge.</li>
		<li>Work quickly for this step. Add 36 mL of cold sterile distilled water directly on top of the pellet and mix once carefully. Add 4 mL of 10x PBS after 20 s to make an isotonic solution. Mix once carefully.</li>
		<li>Spin tube for 5 min at 739 x g and 4 &#176;C (for the removal of RBCs). Neutrophils will be in the white pellet.</li>
		<li>Carefully discard the supernatant. Perform step 1.6.2 again and make sure the pellet is suspended properly.</li>
		<li>Spin tube for 5 min at 328 x g and 4 &#176;C.</li>
		<li>Carefully remove the supernatant and resuspend the pellet in 5 mL of PBS. Count the neutrophils and keep them on ice. 
		NOTE: Expected yield from 1 tube of blood (10 mL) is approximately 15-75 million neutrophils.</li>
	</ol>
	</li>
</ol>
2. Red Fluorescent Cell Labelling of Neutrophils
<ol>
	<li>Make a neutrophil suspension of 10-20 million neutrophils in 2 mL of PBS in a 15 mL tube.</li>
	<li>Make a solution of 2 mL PBS with 4 &#181;L of 2 &#181;M red fluorescent cell linker (see Table of Materials) in a different 15 mL tube. Add this gently to the neutrophil suspension and mix carefully.</li>
	<li>Incubate in the dark for exactly 25 min at 37 &#176;C to label the neutrophils with the red fluorescent cell linker.</li>
	<li>Inactivate the labeling by adding RPMI 1640 medium containing 10% heat inactivated fetal bovine serum (FCS)&#160;at RT up to 15 mL and mix once carefully. Make sure that the pellet is carefully resuspended if a pellet has formed.</li>
	<li>Spin tube for 5 min at 328 x g and RT.</li>
	<li>Remove the supernatant and resuspend pellet in 5 mL of phenol red-free RPMI 1640 medium containing 2% FCS and 10% P/S at RT. Count the neutrophils. 
	NOTE: A cell loss of 50% can occur after red fluorescent cell labelling.</li>
</ol>
3. Induction of Neutrophil Extracellular Trap Formation
<ol>
	<li>Make a cell suspension of 0.42 x 106 cells/mL in phenol red free RPMI 1640 medium containing 2% FCS and 10% P/S.</li>
	<li>Add 37,500 neutrophils in 90 &#181;L per well in a black 96-well, flat bottom plate.</li>
	<li>Add 10 &#181;L of the chosen stimulus (e.g., sera of patient) in triplicate to reach a concentration of 10% in the well. Always include a negative control (medium) in triplicate.</li>
	<li>Incubate in the dark at 37 &#176;C for the desired time, ranging from 30 min to 2, 4 or 6 h. Incubating for 4 h is suggested.</li>
	<li>Calculate the volume needed to add 25 &#181;L of 5 &#181;M impermeable DNA dye (see Table of Materials), to reach a final concentration of 1 &#181;M in the well. Make a predilution if necessary in RPMI 1640 medium containing 2% FCS and 10% P/S to obtain a 5 &#181;M concentration.</li>
	<li>Add 25 &#181;L of 5 &#181;M impermeable DNA dye 15 min before the end of the incubation time. Continue incubation for another 15 min at 37 &#176;C in the dark.</li>
	<li>Remove the supernatant (~125 &#181;L) very carefully and store if needed. Add 100 &#181;L of 4% paraformaldehyde (PFA). Keep in the dark, and immediately continue with step 4.</li>
</ol>
4. NET Visualization with 3D High-content, High-resolution Immunofluorescence Confocal Microscopy
<ol>
	<li>Configure the settings on the immunofluorescence confocal microscope by clicking on the Acquisition setup.
	<ol>
		<li>Click on the Configure tab.</li>
		<li>Select the objective and the camera. Choose the 10X Apo Lambda objective with an acquisition mode of a confocal 60 &#181;m pinhole.</li>
		<li>Click on Plate and choose the 96-well plastic plate. Select the sites to visit and choose a fixed number of sites. Fill in 3 columns and 3 rows without overlap (0 &#181;m), which cover a total of 45% of the well.</li>
		<li>Click on Acquisition. Select Enable Laser-Based Focusing. Select Acquire Z Series/Time Series if needed.</li>
		<li>Click on Site Autofocus.
		<ol>
			<li>Click on Focus on Plate Bottom | Off-Set by Bottom Thickness. For the initial well to find the sample, choose the first well acquired. For the site autofocus, choose all sites.</li>
		</ol>
		</li>
		<li>Click on Wavelengths.
		<ol>
			<li>For the number of wavelengths, choose 2. For the TL shading correction refinement level, choose 2.</li>
			<li>For Wavelength 1, select Texas Red.
			<ol>
				<li>For the autofocus options, select laser with Z offset, post laser offset 1.1 &#181;m. Use Z-stack with a custom range of 200-10.</li>
			</ol>
			</li>
			<li>For Wavelength 2, select FITC.
			<ol>
				<li>For the autofocus options, select laser with Z offset from w1 0 &#181;m. Use Z-stack with a custom range of 200-10.</li>
				<li>For the acquisition options, select Z series and 2D projection image maximum. For the acquisition options, select Shading Correction | Off.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Select Z Series. Select the number of steps: 10. Select the step size: 3 &#956;m (total range will be 27 &#181;m).</li>
	</ol>
	</li>
	<li>Put the plate in the immunofluorescence confocal microscopy.
	<ol>
		<li>Click on the Run tab.
		<ol>
			<li>Fill out plate name and description and choose the storage location.</li>
			<li>Select the wells that need to be acquired.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Choose exposure time for Texas Red and FITC.</li>
	<li>Click on Acquire Plate to start the acquisition, which will take approximately 1 h per plate.</li>
</ol>
5. Analysis of NET Formation
<ol>
	<li>Use an image processing program designed for analysis of scientific multidimensional images (see Table of Materials) to analyze NET formation.</li>
	<li>Transfer the acquired image data to a separate hard drive.</li>
	<li>Select the color adding tool.
	<ol>
		<li>Select w1 and choose the folder in the hard drive where the data is stored.</li>
		<li>Select w2 and choose the folder in the hard drive where the data is stored. 
		NOTE: Use a standard macro that uses w1 in the file name to add red color to the Texas Red images and uses w2 to add green color to the FITC images.</li>
	</ol>
	</li>
	<li>Select Analysis Macro.
	<ol>
		<li>Select w1, choose the threshold value (intensity threshold), which is usually 10. Select the desired pixel value (size threshold, e.g., 100).</li>
		<li>Select w2, choose the threshold value (intensity threshold), which is usually 10. Select the desired pixel value (size threshold, e.g., 500).</li>
		<li>Choose the destination for the spreadsheet file, run analysis, and save log files afterwards.</li>
	</ol>
	</li>
	<li>Analyze data in a spreadsheet.</li>
</ol>

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I., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+quantification+of+in+vitro+NET+formation.">Automatic quantification of in vitro NET formation.</a> Frontiers in Immunology. 3, 413 (2012).</li><li>Nakazawa, D., 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=Enhanced+formation+and+disordered+regulation+of+NETs+in+myeloperoxidase-ANCA-associated+microscopic+polyangiitis.">Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCA-associated microscopic polyangiitis.</a> Journals of the American Society of Nephrology. 25 (5), 990-997 (2014).</li><li>Konig, M. F., Andrade, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Reappraisal+of+Neutrophil+Extracellular+Traps+and+NETosis+Mimics+Based+on+Differential+Requirements+for+Protein+Citrullination.">A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination.</a> Frontiers in Immunology. 7, 461 (2016).</li><li>Kenny, E. F., 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=Diverse+stimuli+engage+different+neutrophil+extracellular+trap+pathways.">Diverse stimuli engage different neutrophil extracellular trap pathways.</a> Elife. 6, (2017).</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> Journal of Immunological Methods. 423, 104-110 (2015).</li><li>Pilsczek, F. H., 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=A+novel+mechanism+of+rapid+nuclear+neutrophil+extracellular+trap+formation+in+response+to+Staphylococcus+aureus.">A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus.</a> The Journal of Immunology. 185 (12), 7413-7425 (2010).</li><li>Tanaka, K., 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=In+vivo+characterization+of+neutrophil+extracellular+traps+in+various+organs+of+a+murine+sepsis+model.">In vivo characterization of neutrophil extracellular traps in various organs of a murine sepsis model.</a> PLoS One. 9 (11), e111888 (2014).</li><li>Fuchs, T. A., 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=Novel+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> Journal of Cell Biology. 176 (2), 231-241 (2007).</li><li>von K&#246;ckritz-Blickwede, M., Chow, O., 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>Kraaij, T., 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=Excessive+neutrophil+extracellular+trap+formation+in+ANCA-associated+vasculitis+is+independent+of+ANCA.">Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA.</a> Kidney International. , (2018).</li><li>Kraaij, T., 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=The+NET-effect+of+combining+rituximab+with+belimumab+in+severe+systemic+lupus+erythematosus.">The NET-effect of combining rituximab with belimumab in severe systemic lupus erythematosus.</a> Journal of Autoimmunity. , (2018).</li><li>Hahn, J., Schauer, C., 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=Aggregated+neutrophil+extracellular+traps+resolve+inflammation+by+proteolysis+of+cytokines+and+chemokines+and+protection+from+antiproteases.">Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.</a> The FASEB Journal. (Aug 21), (2018).</li></ol>

The authors have nothing to disclose.

The most critical part of this assay is the need to use freshly isolated neutrophils for each experiment because neutrophils are short-lived and die when frozen. This requires a healthy donor each time, which could implicate some variations due to donor characteristics. One of these variations is the activation status of the neutrophils. Neutrophils could be activated already in vivo prior to isolation. In addition, neutrophils can be activated throughout the isolation steps notably during lysis of erythrocytes, therefore an experienced handler of neutrophils is required to minimize the activation of neutrophils. In general, isolation of neutrophils should be performed as soon as possible after blood drawing and the experiment should not be paused to avoid excessive spontaneous activation. Secondly, rough handling of neutrophils should be avoided. As such, the notable advantage of the described protocol is the minimal pipetting interventions once neutrophils are seeded in a 96-well plate. Importantly, the activation status of the neutrophils is best assessed in the unstimulated condition, in which this assay can sensitively detect low levels of NET formation. Another factor that could influence the assay is the use of FCS in the medium. The percentage of FCS has been decreased from 10% to 2% to avoid the possible suppression of NET formation by antioxidant activity19,20 or the possible activation of the neutrophils despite heat inactivation. Culture without FCS or with the use different types of media has not been tried. An unstimulated or medium control is always taken along when performing the assay to have an indication of the background signal (e.g., activation status of the neutrophils). The fold increase for each stimulus compared to the unstimulated sample is displayed to achieve consistent results over different experiments using the same stimulus.
An important factor for a possible high background is staining of extracellular DNA that is unrelated to the process of NET formation. The present assay attempts to reduce this by removing the extracellular DNA staining immediately after the short incubation period of 15 min and by analyzing the plate directly after fixation. Therefore, it is essential to use an advanced confocal microscope that has enough speed and analytical power to capture the 96-well plate within 1 to 2 h. Automated setting of the exposure time and focus is recommended. As such, the microscope setting can vary between each sample and experiment with respect to the color intensity threshold which is necessary for overall optimal picture quality. The latter influences the eventual capability to correctly quantify neutrophils and NETs and the optimal setting should therefore be confirmed by using multiple control samples (e.g.,&#160;serum of healthy controls). During the analysis of captured images, the use of a pixel threshold and size threshold in the analysis program allows for a better selection of NET formation.
Extracellular DNA derived from NET formation can be the result of distinct death pathways, including NETosis, necroptosis, pyroptosis, ferroptosis or even non-lytic process of vital NET formation1. As such, a limitation of the present assay is that by staining only for extracellular DNA there is no differentiation possible between NET formation and other relevant cell death pathways. It is possible to achieve this by using either selective inhibitors of distinct death pathways to discriminate between the different forms underpinning NET formation or confirm by separate immunostainings the presence of specific NET markers, such as citH3 and NE, co-localized with DNA. The co-localization of extracellular DNA with citH3 and NE has recently been confirmed for this assay10. The advantage of avoiding NET specific markers in this assay, allows to assess all forms of NET formation leading to the extrusion of DNA by neutrophils as complete and as objective as possible with the potential of high-throughput screening. The applicability of this protocol has been shown in studying low level NET induction mediated by immune complexes in auto-immune disease in which the ability to detect qualitative and quantitative differences might be more important than the type of process involved10,21,22. Illustrating that this novel NET quantification assay can be of added value for different researchers to investigate various aspects of NET formation. Small adjustments to the assay are easily implemented: adjustment of the stimulation period, the use of a favorite NET marker to focus on one specific death pathway leading to NET formation, the use of a different magnification or the use of different NET criteria in the quantification and analysis.
In conclusion, the protocol provided is a highly-sensitive broadly applicable assay for the semi-automated quantification of NET formation for the evaluation of ex vivo induction of NETs upon different stimuli.

The formation of neutrophil extracellular traps (NETs) is the process in which neutrophils release their DNA in an extracellular three-dimensional (3D) web like structure, complexed with a wide range of antimicrobial and dangerous molecules, granular and cytoplasmic enzymes, peptides and proteins. These immunogenic and toxic structures have an important physiological role in the innate immune defense of healthy individuals by trapping and killing infectious pathogens1. However, they have also been demonstrated to be involved in thrombosis2 and various systemic autoimmune diseases, including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV)3, systemic lupus erythematosus (SLE)4,5, antiphospholipid syndrome (APS)2,6, rheumatoid arthritis (RA), psoriasis, and gout7,8,9.
In vitro NET formation has been widely studied with the chemical compound phorbol 12-myristate 13-acetate (PMA), which induces massive NET formation. However most physiological stimuli induce much lower levels of NET formation10. To study NET-triggers in, for example, an autoimmune disease setting, a standardized, sensitive, high-throughput quantitative assay is needed to detect and quantify NET formation. Quantification of NETs has proven to be challenging and is currently performed by different methods, each with their own advantages and limitations11. A commonly used method is the detection of DNA in the supernatants12, which is objective but does not discriminate between the origin of the DNA (apoptotic, necrotic, NETs), and therefore is not very specific for NETs. Secondly, enzyme-linked immunosorbent assays (ELISAs) of DNA-complexed with NET-specific proteins, for example, myeloperoxidase (MPO) or neutrophil elastase (NE), are a more specific approach to detect NETs and were demonstrated to correlate well with citrullinated histone-3 (CitH3) positive NETs13. However, it is not known whether this method is sensitive enough to pick up all NETs (e.g.,&#160;MPO, NE, and CitH3 negative NETs). A third approach is immunofluorescence microscopy that is used to detect co-localization of NET-associated molecules (NE, MPO, CitH3) with extracellular DNA to quantify NETs. This method is generally specific for NETs, but it cannot be applied as a high-throughput method and is not objective due to observer bias. Additionally, this method does not take into account MPO-, NE-, CitH3-negative NETs that are frequently present depending on the used NET-trigger14,15. Flow cytometry approaches detect NETs through a changed forward/sideward scatter (FSC/SSC) indicating swelling of the nucleus in NET-ting neutrophils16. This method does not take into account the different forms of NET formation that have been identified, which might not involve swelling of the nucleus, such as vital NET formation17. Lastly, immunofluorescence confocal microscopy has been applied to visualize and quantify NET formation by directly staining extracellular DNA with a cell-impermeable dye that stains extracellular DNA12,18. Generally, 5 to 10 high-power fields are manually picked and assessed, which covers 1-5% of each well of a 96-well plate11,17. Manual selection of images is not always objective, prone to bias and not attractive for high-throughput analysis. An automated, high-throughput NET quantification assay was recently developed, which imaged 11% of the well in a 3D manner covering 13 &#181;m through Z-stacked immunofluorescence confocal microscopy, thereby leading to a highly sensitive technique to assess NETs compared to the conventional methods10. The current report describes the most recent protocol to quantify NET formation through an automated, highly sensitive assay using 3D confocal microscopy, which achieves a total imaged area of 45% of each well and covers 27 &#181;m through Z-stacks. This protocol is suitable to quantify, with a high sensitivity, low levels of NET formation in an objective and unbiased manner.

<table><tbody><tr><td>Aqua Sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td>B. Braun, Melsungen, Germany</td><td>12604052</td><td></td></tr><tr><td>Fetal bovine serum (FCS)</td><td>Bodinco, Alkmaar, The Netherlands</td><td></td><td>Used in high concentrations it could influcence NET formation</td></tr><tr><td>Ficoll 5.7% - amidotrizoaat 9% density 1.077 g/mL</td><td>LUMC, Leiden, The Netherlands</td><td>97902861</td><td></td></tr><tr><td>Immunofluorescence confocal microscope</td><td>Image Xpress Micro Confocal (Molecular Devices, Sunnyvale, CA, USA)</td><td></td><td></td></tr><tr><td>Neutralization PBS (10x)</td><td>Gibco, Paisley, UK</td><td>70011-036</td><td></td></tr><tr><td>Penicillin/streptomycin (p/s)</td><td>Gibco, Paisley, UK</td><td>15070063</td><td></td></tr><tr><td>Phenol red free RPMI 1640 (1x)</td><td>Gibco, Paisley, UK</td><td>11835-063</td><td>Phenol red can interfere with the immunofluorescence signal</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>B. Braun, Melsungen, Germany</td><td>174628062</td><td></td></tr><tr><td>PKH26 2 &amp;mu;M Red fluorescent cell linker</td><td>Sigma Aldrich Saint-Louis, MO, USA</td><td>PKH26GL-1KT</td><td>PKH are patented fluorescent dyes and a cell labeling technology named after their discoverer Paul Karl Horan</td></tr><tr><td>Program for scientific multidimensional images analysis</td><td>ImageJ, Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA</td><td></td><td></td></tr><tr><td>RPMI medium 1640 (1x)</td><td>Gibco, Paisley, UK</td><td>52400-025</td><td></td></tr><tr><td>Sytox green 5 mM Impermeable DNA dye</td><td>Gibco, Paisley, UK</td><td>57020</td><td></td></tr><tr><td>Trypan blue stain 0.4%</td><td>Sigma Aldrich, Germany</td><td>17942E</td><td></td></tr><tr><td>96 well, black, flat bottom, tissue culture treated plate</td><td>Falcon, NY, USA</td><td>353219</td><td></td></tr></tbody></table>

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.

Neutrophil extracellular trap (NET) formation is quantified in a 3D manner by quantifying stained extracellular DNA over 10 Z-stacks with 3 &#181;m distance starting off at the focal plane in each well. By measuring the cumulative area, the sensitivity of the assay increases (Figure 1A). The isolated neutrophils have a mean purity of 98.7% with standard deviation (SD) of 1.10% measured in 14 different samples from different isolations. The mean percentage of red blood cells is 1.04% &#177; 1.1% SD and the mean percentage of monocytes is 0.085% &#177; 0.17% SD (data not shown). The total area of stained neutrophils imaged are quantified only in the focal plane in each well, which correlate significantly with the total neutrophil count in each well with a Pearson correlation coefficient of 0.99 (95% confidence interval [CI] 0.985-0.997, p&#160;&#60; 0.0001) (Figure 1B). The representative outcome of quantification of NET formation in neutrophils stimulated with 10% sera of AAV patients or medium (MED) as a negative control, expressed as cumulative stained extracellular DNA area over 10 Z-stacks per imaged neutrophil (Figure 1C). Snapshots of representative images of unstimulated neutrophils (Figure 2A) and of NETs in AAV-stimulated neutrophils (Figure 2B) are shown.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59150/59150fig1.jpg" /> 
Figure 1: Quantification of NET formation by measuring extracellular DNA and neutrophil count. (A)&#160;Area is cumulatively quantified over the 10 Z-stacks for each well for unstimulated neutrophils (MED) and for neutrophils stimulated with 10% serum of ANCA-associated vasculitis (AAV) patients (n = 4) quantified with an image processing program. Each stimulus was tested in triplicate, each point represents the median value. (B) Red fluorescent labelled cell area and cell count were quantified in the focal plane of each well by the image processing program (R2 = 0.99, p&#160;&#60; 0.0001). (C)&#160;NET formation is expressed as cumulative area per imaged neutrophil (cell area). The mean &#177; standard error of the mean (SEM) of each triplicate is plotted per stimulus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59150/59150fig1large.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/59150/59150fig2.jpg" /> 
Figure 2: Snapshots of NET quantification assay. Fluorescent labelled neutrophils are shown in red, and stained extracellular DNA is shown in green. 10x Plan Apo Lambda objective. (A) Unstimulated neutrophils. (B) Neutrophils stimulated with AAV serum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59150/59150fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation at JoVE.com

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

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. ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

10.0K Views.  Leiden University Medical Center. We ontwikkelden een high-throughput test om neutrofiele extracellulaire vallen te meten, of NETs. NETs zijn immunogene DNA-structuren die worden blootgesteld door neutrofielen. NETs kunnen micro-organismen vangen en doden en zijn daarom een belangrijk antisinstekelijk mechanisme, maar ze spelen ook een pathogene rol bij auto-immuunziekten.Met deze techniek worden NETs's gekwantificeerd door driedimensionale immunofluorescentieconfocale microscopie wat resulteert in een zeer gevoelige e...

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

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 ...

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

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 ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

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

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 ...

Neutrophil Functional Assays for Screening Different Signaling Pathways

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

Author Spotlight: Balancing Speed, Cost, and Accuracy in Neutrophil Function Assessment with NeutroFun Screen Protocol 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

Real-Time Microscopy for Studying NETosis Kinetics and Screening Inhibitors

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

This defined protocol describes a real-time high-throughput microscopy approach to visualize and quantify human neutrophil extracellular trap (NET) release in vitro. The reproducible method allows investigation of the characteristics and kinetics of NET release upon stimulation with distinct NETosis inducers and enables assessment of the pharmacology of NETosis antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in ...

Biology

Induction and Visualization of Neutrophil Extracellular Traps in Pre-Labeled Neutrophils

Source: Arends, E. J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59150">A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates the induction and visualization of neutrophil extracellular traps or NETs in pre-labeled neutrophils using patient serum containing anti-neutrophil cytoplasmic antibodies. Furthermore, using an impermeable DNA dye and fluorescence microscopy enables the visualization of NETs.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host Defense Mechanism

An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

To stimulate the formation of neutrophil extracellular traps, or NETs, add 500 nanomolar of PMA to 30 milliliters of the neutrophil suspension in a 150-millimeter by 25-millimeter flat tissue culture dish with a 20-millimeter grid. Incubate the cells for four hours at 37 degrees Celsius under 5% carbon dioxide.
After incubation, gently aspirate and discard the medium, taking care not to disrupt the layer of NETs and neutrophils adhered to the bottom of the dish. Then, use a pipette to wash the bottom of each dish with 15 milliliters of cold PBS without calcium and magnesium. All adherent material should be removed from the bottom.
Collect the wash suspension in a 15-milliliter conical tube and then centrifuge at 450 x g for 10 minutes at 4 degrees Celsius. Neutrophils and any remaining cells will pellet at the bottom, leaving a cell-free, NET-rich supernatant.
Next, decant the supernatant into multiple 1.5-milliliter tubes and centrifuge at 18,000 x g for 10 minutes at 4 degrees Celsius to pellet the DNA. Discard the supernatant, and re-suspend the pellet in PBS such that the concentration corresponds to 2 x 107 neutrophils per 100 microliters of PBS. This cell-free NET stock can be used for subsequent experiments.

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

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