The authors thank Dr. Huq Muhammad Aminul for providing assistance in reviewing the manuscript.

This study was conducted in conformity with the Declaration of Helsinki and was approved by the institutional review boards of Aichi Medical University (2017-H341, 2019-H137). Written informed consent was obtained from each participant.
1. Reagent preparation
NOTE: To perform the sandwich ELISA assay, the reagents are prepared as described below.
<ol>
	<li>Coating buffer:
	<ol>
		<li>To make a 0.1 mol/L carbonate-bicarbonate buffer, weigh 10.6 g of anhydrous sodium carbonate (molecular weight, 106 g/mol) and 8.4 g of sodium bicarbonate (molecular weight, 84 g/mol).</li>
		<li>Add it to approximately 900 mL of distilled water in a beaker.</li>
		<li>Adjust the pH of the buffer to 9.6 by adding the required quantity of diluted hydrochloric acid or sodium hydroxide.</li>
		<li>Check the pH with a pH meter.</li>
		<li>Then, add the required volume of distilled water to achieve a total volume of 1,000 mL.</li>
		<li>Store this 0.1 mol/L carbonate-bicarbonate buffer in the refrigerator at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Blocking buffer:
	<ol>
		<li>Add approximately 250 &#181;L of 20% sodium azide and 1 g of bovine serum albumin (BSA) to 70 mL of phosphate-buffered saline (PBS) composed of 137 mmol/L NaCl, 8.1 mmol/L Na2HPO4, 2.68 mmol/L KCl, and 1.47 mol/L KH2PO4 (pH 7.4), and mix gently.</li>
		<li>Add distilled water to achieve a total volume of 100 mL. The final concentrations of bovine serum albumin and sodium azide are 1% and 0.05%, respectively. Wait for 10 min, and then the solution is ready to use.</li>
	</ol>
	</li>
	<li>Washing solution: Prepare 0.5% t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether (Triton X-100) in distilled water.
	<ol>
		<li>Dilute 100% Triton X-100 to 10% with distilled water, and allow the 10% solution to stand for 1 day before use.</li>
		<li>Then, re-dilute the 10% Triton X-100 solution 20-fold with distilled water to achieve a concentration of 0.5%.</li>
	</ol>
	</li>
	<li>Antibody dilution:
	<ol>
		<li>Dilute the capture antibody (anti-MPO antibody [1 mg/mL] or anti-NE antibody [1 mg/mL]) to 1:2,000 in the coating buffer (pH 9.6) before use on day 1 of the assay (see below) and the detection antibody (peroxidase-conjugated anti-DNA antibody) to 1:40 with PBS before use on day 3 of the assay (see below). 
		NOTE: For the preliminary experiment, dilute the iso-type antibodies (IgG rabbit [5 mg/mL] and IgG1 mouse clone Ci4 [0.5 mg/mL]) to 1:10,000 and 1:1,000 in the coating buffer (pH 9.6).</li>
	</ol>
	</li>
	<li>ABTS substrate solution: Depending on the number of samples, dissolve one, two, or three 2,2&#39;-azino-bis(3 ethylbenzothiazoline-6-sulfonic acid (ABTS) tablets in 5 mL, 10 mL, or 15 mL of ABTS buffer (containing sodium perborate, citric acid, and disodium hydrogen phosphate); 100 &#181;L is needed per sample. Store the prepared solution at 2-8 &#176;C protected from light. The solution is stable for up to 3 months.</li>
</ol>
2. Sample collection and storage
<ol>
	<li>Plasma isolation:
	<ol>
		<li>Collect 4 mL of peripheral blood samples into a lithium heparin blood collection tube by venipuncture with a 22 G syringe, and centrifuge at 1,600 x g for 10 min at 4 &#176;C.</li>
		<li>Transfer the supernatant into a 2 mL safe-lock sample tube.</li>
		<li>Re-centrifuge the supernatant at 16,000 x g for 10 min at 4 &#176;C to remove any residual cells.</li>
		<li>Collect the supernatant into a new safe-lock sample tube without disturbing the pellet at the bottom of the tube.</li>
		<li>Store the obtained plasma at &#8722;80 &#176;C until further use. 
		NOTE: To obtain high-throughput results with this assay, good-quality samples are required. If plasma samples are used, perform a second centrifugation to remove any residual cells. Avoid repeated thawing of the samples. Use heparin as the anticoagulant because EDTA affects the DNase activity.</li>
	</ol>
	</li>
	<li>Isolation of polymorphonuclear neutrophils
	<ol>
		<li>Collect the peripheral blood samples into a lithium heparin blood collection tube by venipuncture with a 22 G syringe.</li>
		<li>Layer 6 mL of blood onto 6 mL of one-step polymorphs, and centrifuge the tubes at 1,000 x g for 45 min at room temperature (RT).</li>
		<li>Harvest the third buffy coat layer containing neutrophils, and wash it three times in phenol red-free RPMI-1640 at 400 x g for 10 min at RT.</li>
		<li>Resuspend the neutrophils in phenol red-free RPMI-1640 containing 2 mM L-glutamine supplemented with 6% heat-inactivated fetal bovine serum, and count the cells in the microscope field with a hemocytometer. 
		NOTE: This method yields 96% to 98% purity with greater than 95% viability, as assessed by trypan blue dye exclusion.</li>
	</ol>
	</li>
	<li>Activation of polymorphonuclear neutrophils and NETosis in vitro
	<ol>
		<li>Dilute freshly isolated polymorphonuclear neutrophils to 1 &#215; 105 cells/mL in phenol red-free RPMI-1640 containing 2 mM L-glutamine supplemented with 6% heat-inactivated fetal bovine serum, and seed them in 35 mm culture dishes.</li>
		<li>To induce NETs, stimulate polymorphonuclear neutrophils with 25 nM PMA for 4 h at 37 &#176;C in 5% CO2/95% air.</li>
		<li>After 4 h of incubation, partially digest the NETs by adding 0.6 &#181;g/mL DNase I directly to the culture dishes for 15 min at RT, and stop the DNase I activity by adding 5 mM EDTA.</li>
		<li>Collect the medium containing the synthesized NETs, and centrifuge at 400 x g for 10 min at 4 &#176;C to remove the cell debris.</li>
		<li>Obtain the supernatants from four healthy controls, mix, and store at &#8722;80 &#176;C until use. 
		&#8203;NOTE: DNase-digested PMA-stimulated neutrophils obtained from four healthy controls are used as the NET-standard1,14,15. Generate a calibration curve from a serially diluted NET-standard with PBS, and assign the optical density (OD) values obtained from an undiluted NET-standard as 100% NET.</li>
	</ol>
	</li>
</ol>
3. Assay method
NOTE: The steps for performing the assay are described in detail below.
<ol>
	<li>Day 1
	<ol>
		<li>Apply a total of 100 &#181;L of diluted anti-MPO or anti-NE antibody containing 0.05 &#181;g of antibody to each well of the plate, including the blank well. 
		NOTE: The coating buffer must not contain any kind of detergent because, otherwise, the antibodies may not bind equally and smoothly to the walls of each well. To prevent the hook effect, the coating protein concentration must not be more than 20 &#181;g/mL, because concentrations above that level will saturate most of the available sites on the microtiter plate. The typical concentration range of protein coating solutions is 2-10 &#181;g/mL. For the preliminary experiment, use diluted iso-type control antibody IgG rabbit for coating instead of specific monoclonal antibodies against MPO. Use diluted iso-type control antibody IgG1 mouse for coating instead of specific monoclonal antibodies against NE.</li>
		<li>Cover the plate with an adhesive plastic cover to prevent the evaporation of the sample, and incubate overnight at 4 &#176;C to allow the binding of the capture antibodies.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Discard the diluted antibody solution from the wells, and then pipette 300 &#181;L of wash solution per well, and repeat this washing procedure three to four times.</li>
		<li>Remove excess PBS by tapping the plate dry on a paper towel.</li>
		<li>Block each well of the ELISA plate with 200 &#181;L of the blocking buffer.</li>
		<li>Cover the plate closely with an adhesive plastic cover, and incubate it for 1.5-2 h at RT to obstruct the wells.</li>
		<li>Discard the blocking solution completely from the wells, and then pipette 300 &#181;L of wash solution per well, and repeat this washing procedure three to four times.</li>
		<li>Remove excess PBS by tapping the plate dry on a paper towel.</li>
		<li>Apply a total of 25 &#181;L of plasma or medium to each well except the blank well, and add 75 &#181;L of PBS as a diluent to make the final volume 100 &#181;L; add 100 &#181;L of PBS to the blank well.</li>
		<li>Then, mix the samples at RT for 10 s by placing the plate on a shaker at 250 rpm.</li>
		<li>Apply 2 &#181;L of 100-fold diluted DNase I (0.03 mg/mL) to each well, including the blank well (final concentration of DNase I in the reaction mixture: 0.6 &#181;g/mL).</li>
		<li>Seal the plate with an adhesive plastic cover, and thoroughly mix the samples at RT for 10 s by placing the plate on a shaker at 250 rpm. 
		NOTE: To assess the optimum conditions for DNA digestion, when developing the assay, we incubated samples containing MPO-associated DNA from patients with COVID-19 with 0-0.9 &#181;g/mL DNase I for 15 min at RT and used plates coated with specific capture antibody&#160;for MPO or a species-matched iso-type control antibody.</li>
		<li>Incubate the samples for 15 min at RT.</li>
		<li>Remove the adhesive plastic cover, and apply 1 &#181;L of 0.5 M EDTA to each well to stop the DNase reaction.</li>
		<li>Reseal the plate with an adhesive plastic cover, and shake it at RT for 15 s by placing it on a shaker at 250 rpm.</li>
		<li>Then, incubate the plate overnight at 4 &#176;C to allow the protein components of the NETs to attach to the capture antibodies.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Remove the adhesive plastic cover, and discard the solution from the wells.</li>
		<li>Discard the solution completely from the wells, and then pipette 300 &#181;L of the wash solution per well, and repeat this washing procedure three to four times.</li>
		<li>Apply a total of 100 &#181;L of the diluted peroxidase-conjugated anti-DNA detection antibody to each well.</li>
		<li>Seal the plate closely with an adhesive plastic cover, and incubate it for 1.5 h at RT.</li>
		<li>Discard the solution completely from the wells, and then pipette 300 &#181;L of wash solution per well, and repeat this washing procedure three to four times.</li>
		<li>Carefully remove the residual solution.</li>
		<li>Apply a total of 100 &#181;L of ABTS substrate solution to each well, and cover the plate with an adhesive plastic cover.</li>
		<li>Incubate the plate in the dark at RT for 20-30 min on a shaker at 250 rpm. 
		NOTE: Monitor the plate at 5 min intervals until the desired OD readings are obtained.</li>
		<li>Add a total of 50 &#181;L of 2 M sulfuric acid to stop the reaction.</li>
		<li>Mix the liquid contents of the wells by carefully tapping the side of the plate.</li>
		<li>Switch on the microplate reader, and connect it to the computer.</li>
		<li>Open the software application on the computer.</li>
		<li>From the status bar, create a new experiment and name it.</li>
		<li>Set all the following parameters for plate reading: Read type, Absorbance; Read mode, Endpoint; Wavelengths, 2; Lm-1, 405 nm; Lm-2, 490 nm; Auto mix &#38; Blanking before, Off; Pre-read plate, Off; Auto calibration, On; Strips, Read entire plate; Column wavelength priority, Column priority; Carriage speed, Normal; and Auto read, Off.</li>
		<li>Then, put the 96-well plate containing the samples into the drawer and close it.</li>
		<li>Last, select the Read button so that the plate is read immediately.</li>
		<li>Read the absorbance of each well at a wavelength of 405 nm by using a microplate reader (at this stage, use a wavelength of 490 nm as a reference).</li>
		<li>Perform automatic subtraction of the absorbance value of the assay medium alone from all the unknown samples, and save the data. 
		&#8203;NOTE: Use a standard curve obtained from serially diluted NET-standard to calculate the relative concentration of NET in the sample1,14,15. Here, the final results are presented as &#34;MPO- or NE-DNA complexes (% of NET-standard).&#34; The limit of detection (LOD) was calculated from the standard deviation (SD) and the slope of the calibration curve (S) as follows: LOD = 3.3(SD/S).</li>
	</ol>
	</li>
</ol>
4. Statistics
<ol>
	<li>Perform all the statistical analyses using appropriate statistical analysis software. Here SigmaPlot v14.5 was used. Percentages and continuous variables are shown as the median with the interquartile range due to the skewed distribution of most of the parameters.</li>
	<li>Compare the plasma MPO-DNA and NE-DNA levels between COVID-19 patients and healthy controls using the Wilcoxon rank sum test. Use correlation analysis to explore the relationship between optical density and the percentage of NET-standard.</li>
</ol>

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<li>Prevel, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasma+markers+of+neutrophil+extracellular+trap+are+linked+to+survival+but+not+to+pulmonary+embolism+in+COVID-19-related+ARDS+patients%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Plasma markers of neutrophil extracellular trap are linked to survival but not to pulmonary embolism in COVID-19-related ARDS patients.</a> Frontiers in Immunology. 13, 851497(2022).</li>
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<li>Li, M., Lin, C., Leso, A., Nefedova, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+citrullinated+histone+H3+bound+DNA+for+detection+of+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22Cancers%22%5BJournal%5D)">Quantification of citrullinated histone H3 bound DNA for detection of neutrophil extracellular traps.</a> Cancers. 12 (11), 3424(2020).</li>
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<li>Zuo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+in+COVID-19%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Neutrophil extracellular traps in COVID-19.</a> JCI Insight. 5 (11), e138999(2020).</li>
<li>Masso-Silva, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+peripheral+blood+neutrophil+activation+phenotypes+and+neutrophil+extracellular+trap+formation+in+critically+ill+Coronavirus+disease+2019+%28COVID-19%29+patients%3A+A+case+series+and+review+of+the+literature%5BTitle%5D)+AND+%22Clinical+Infectious+Diseases%22%5BJournal%5D)">Increased peripheral blood neutrophil activation phenotypes and neutrophil extracellular trap formation in critically ill Coronavirus disease 2019 (COVID-19) patients: A case series and review of the literature.</a> Clinical Infectious Diseases. 74 (3), 479-489 (2022).</li>
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</ol>

All the data generated and/or analyzed during this study are included in this published article. The authors declare that they have no competing interests.

We have described a sandwich ELISA method in which MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. After washing, the &#34;sandwich&#34; is completed by incubating the samples with a peroxidase-associated anti-DNA monoclonal antibody. After the removal of unbound secondary antibody, the bound peroxidase conjugate is detected by the addition of a chromogenic ABTS peroxidase substrate, which yields a soluble end-product that can be read spectrophotometrically at 405 nm. The good linearity and high inter-assay and intra-assay precision indicate that the ELISA assay described in this paper and others1,2&#160;is reliable and feasible for clinical application. Furthermore, when the supernatant of DNase-treated PMA-stimulated neutrophils is assigned as a maximal signal for the NET-standard, this ELISA assay can be used as a semi-quantitative method1,14,15.
The long chromatin threads of NETs are decorated with MPO and NE proteins. To increase the binding between the capture antibody and the MPO-DNA or NE-DNA complexes, the threads are cut into shorter pieces by limited DNA digestion with the enzyme DNase; the addition of a DNase concentration that is too high could lead to excessive digestion of the DNA and, thus, a decrease in the absorbance. The results of a preliminary experiment (see Figure 3) showed that an appropriate amount of DNase addition is necessary to loosen the remnants derived from NETs. The experiments that used iso-type control antibodies instead of specific capture antibodies showed that the capture antibodies used in this assay were specific for MPO-DNA and NE-DNA complexes.
The early stage of NET formation is characterized by decondensed chromatin and the preservation of the intensity of the plasma membrane9,16,17. Neutrophils with a condensed nucleus have been identified as neutrophils that are undergoing NET formation and can be quantified by flow cytometry10. Although flow cytometry can analyze a large number of images from cells in a short time, it cannot be used to evaluate the later stages of NET formation after cell membrane rupture and the extrusion of chromatin. Citrullinated histones H3, which are known to be NET-specific markers, can be detected by western blotting18 and ELISA19; however, these methods are only specific to PAD4-related NET formation and cannot be used to assess PAD4-independent NETs20.
The finding that the levels of serum NET remnants were higher in patients with COVID-19 than in healthy controls agrees with previous reports21,22. This finding suggests that neutrophil activation, including NET formation, may play an important role in the pathogenesis of COVID-19.
This assay has a limitation. Recent reports have shown that immune-related genes, including MPO and ELANE, which encode MPO and NE, respectively, are highly expressed under uncontrolled inflammatory conditions23 and are positively correlated with disease severity and mortality24. As MPO and NE are involved in the formation of NETs independent of those enzymatic activities15, increased protein levels of NE and MPO in neutrophils may affect the results of this assay.
We conclude that this sandwich ELISA method directly measures extracellular DNA with granule proteins, including NE and MPO, which are specific for NET formation. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human samples and culture supernatants.

This article outlines a method to quantify neutrophil extracellular trap (NET) formation in biological fluids by using sandwich enzyme-linked immunosorbent assay (ELISA) to detect complexes of myeloperoxidase (MPO) and neutrophil elastase (NE) with DNA1,2. NETs are composed of a DNA backbone decorated with antimicrobial proteases originating from neutrophil granules3,4. Both MPO-DNA and NE-DNA complexes are important and specific components of NETs and are released into the extracellular space as breakdown products of NETs3,4. 
Besides their important physiological role in antimicrobial defense3, NETs also have various pathological effects4,5, including the promotion of thrombogenesis6 and the worsening of sepsis7. Accordingly, NETs have been gaining attention recently. Nevertheless, the in vivo quantification of NETs has proven challenging because of the lack of a sensitive, reliable quantitative assay method. 
A few methods are available, including the direct measurement of NETs by fluorescence microscopy8,9 and flow cytometry10 and the indirect measurement of circulating cell-free DNA, nucleosomes, and citrullinated histone H3, but each method has its own advantages and limitations11. Although the immunofluorescence microscopic method is specific to NETs and clearly shows the localization and degree of NET formation, samples are limited to biopsy tissue and secreted materials. Moreover, this method needs to be performed by skilled researchers and requires a long time for results to be obtained. Measuring circulating levels of NET-related components by flow cytometry is easy and provides results quickly; however, the method is not specific to NETs12.
We13 and others1,2 have developed a highly sensitive and reliable assay to measure the circulating NET components, MPO-conjugated or NE-conjugated DNA, in human plasma with a modified ELISA technique that uses specific antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. This assay can also be used ex vivo to identify NET components in cell culture supernatants released by activated neutrophils in response to phorbol 12-myristate 13-acetate (PMA) stimulation.

<table><tbody><tr><td>1-Step Polymorphs</td><td>Accurate Chemical and Scientific Corporation</td><td>AN221725</td><td>Isolation of PMN&#039;s from human blood.</td></tr><tr><td>96-well microtiter plate</td><td>Thermo Fisher Scientific</td><td>467466</td><td>flat bottom</td></tr><tr><td>ABTS buffer solution</td><td>Sigma-Aldrich Merck</td><td>11 204 530 001</td><td>Contains sodium perborate, citric acid, and disodium hydrogen phosphate.&amp;nbsp;</td></tr><tr><td>ABTS tablets</td><td>Sigma-Aldrich Merck</td><td>11 204 521 001&amp;nbsp;</td><td>Each tablet contains 5 mg ABTS substrate and 60 mg vehicle substances.</td></tr><tr><td>Adhesive plastic cover, Axygen</td><td>Thermo Fisher Scientific</td><td>14222348</td><td></td></tr><tr><td>Anti-MPO antibody</td><td>Sigma-Aldrich Merck</td><td>&amp;nbsp;07-496-I</td><td>Store at 2-8 &amp;deg;C. stable for 1 year. Host species is rabbit.</td></tr><tr><td>Anti-NE antibody, clone AHN-10</td><td>Sigma-Aldrich Merck</td><td>MABS461</td><td>Store at 2-8 &amp;deg;C. stable for 1 year. Host species is mouse.</td></tr><tr><td>Bovine serum albumin</td><td>Biomedical Science</td><td>BR-220700081</td><td>Albumin from bovine fraction V. Store at 2&amp;ndash;8 &amp;deg;C. stable for 2 year.</td></tr><tr><td>DNase I</td><td>New England BioLabs</td><td>M0303M</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>IgG, rabbit, Isotype Control</td><td>GENETEX, Inc.</td><td>GTX35035</td><td>Store as concentrated solution at 2&amp;ndash;8 &amp;deg;C.</td></tr><tr><td>IgG1, mouse Isotype Control, clone Ci4</td><td>Merck&amp;nbsp;</td><td>MABC002</td><td>Store as concentrated solution at 2&amp;ndash;8 &amp;deg;C.</td></tr><tr><td>Lithium heparin blood collection tube</td><td>Becton Dickinson and Company</td><td></td><td></td></tr><tr><td>Microplate mixer</td><td>As one corporation</td><td>NS-P</td><td></td></tr><tr><td>Microplate Reader</td><td>Molecular Devices</td><td>SpectraMax 190&amp;nbsp;</td><td>Any microplate plate reader capable of reading wavelengths from 405&amp;ndash;490 nm can use.</td></tr><tr><td>Microplate reader application</td><td>Molecular Devices</td><td>SoftMax pro</td><td></td></tr><tr><td>Peroxidase-conjugated anti-DNA antibody, Cell death Detection ELISA</td><td>Roche Diagnostics</td><td>1154467500</td><td>&amp;nbsp;bottle 2. Store at 2&amp;ndash;8 &amp;deg;C. stable for 1 year.</td></tr><tr><td>Phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich Merck</td><td>P8139</td><td>Activation of PMN&#039;s from human blood.</td></tr><tr><td>Phosphate buffered solution</td><td>Takara Bio</td><td>T9181</td><td>Store at room temperature. Stable for 6 months.</td></tr><tr><td>SigmaPlot v14.5</td><td>&amp;nbsp;Systat Software Inc. San Jose, CA, USA</td><td></td><td></td></tr><tr><td>Sodium azide</td><td>Fujifilm Wako Chemicals</td><td>190-14901</td><td>Store at room temperature.</td></tr><tr><td>t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether</td><td>Fujifilm Wako Chemicals</td><td>9002-93-1</td><td>Store at room temperature.</td></tr></tbody></table>

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.

This method used a sandwich ELISA with anti-MPO, anti-NE, and anti-DNA monoclonal antibodies to measure MPO-associated and NE-associated DNA (Figure 1). In this method, the wells of a microtiter plate were coated with an MPO-specific or NE-specific monoclonal antibody to capture DNA-associated MPO and DNA-associated NE, as well as non-DNA-associated MPO and NE. To calculate the intra-assay coefficient of variability (CV), duplicate measurements were performed within the same plate for 30 samples collected from patients with COVID-19 and healthy controls, and the %CV was calculated as the mean of the duplicate measurements; to calculate the inter-assay CV (i.e., the plate-to-plate consistency), two types of samples from patients with COVID-19 and healthy controls were measured in quadruplicate on 10 different plates; to demonstrate the specificity of this assay, various concentrations of MPO-DNA and NE-DNA complexes were assayed by using plates coated with iso-type control antibodies instead of specific monoclonal antibodies against MPO and NE; and to calculate the sensitivity and linearity of the assay, a serially diluted NET-standard made by stimulating isolated human neutrophils with PMA and mild DNase digestion was assayed, and the correlation coefficient and limit of detection (LOD) were calculated.
The calibration curves for MPO-DNA and NE-DNA drawn from a serially diluted NET-standard are shown in Figure 2. Reliable standard curves for MPO-DNA and NE-DNA (R2 = 0.958 and 0.963, respectively) are obtained when the absorbance values do not exceed concentrations of 0.93 and 0.90, respectively. The LOD values calculated from the SD and the slope of the calibration curve were 0.132% and 0.126% for MPO-DNA and NE-DNA (%NET standard), respectively.
The highest OD was obtained when 0.6 &#181;g/mL DNase I was applied (Figure 3). Therefore, the DNA digestion was limited to adding a 0.6 &#181;g/mL reaction mixture of DNase I for 15 min at room temperature. When the plates were coated with the iso-type control antibody instead of the specific monoclonal antibody against MPO, very low OD values (&#60;0.09 absorbance units [AU]) were detected at various concentrations of DNase I.
To calculate the intra-assay coefficient of variability (CV) for MPO-DNA and NE-DNA, duplicate measurements were performed in 30 samples from patients with COVID-19 and healthy controls in the same plate; the %CV was calculated by using the duplicate mean. The intra-assay CVs for MPO-DNA and NE-DNA were 1.871 and 0.987, respectively, in healthy controls and 2.532 and 2.010, respectively, in COVID-19 patients (Table 1). The mean intra-assay CVs of MPO-DNA and NE-DNA were 2.202 &#177; 0.467 and 1.497 &#177; 0.723, respectively (mean &#177; SD) (Table 1).
To calculate the inter-assay CV for MPO-DNA and NE-DNA, two types of samples collected from patients with COVID-19 and healthy controls were measured in quadruplicate on 10 different plates to monitor the plate-to-plate consistency. The mean inter-assay CVs of MPO-DNA and NE-DNA were 6.524 &#177; 2.672 and 4.389 &#177; 0.923, respectively (mean &#177; SD) (Table 2).
To evaluate the specificity of the capture antibodies to MPO-DNA and NE-DNA complexes, we assayed various concentrations of MPO-DNA and NE-DNA complexes by using plates coated with iso-type control antibodies instead of specific monoclonal antibodies against MPO and NE. Table 3 shows that the iso-type control antibodies reacted little with the MPO-DNA and NE-DNA complexes at the various concentrations (0.035 AU to 0.078 AU and &#8722;0.007 AU to 0.096 AU, respectively).
The levels of MPO-DNA and NE-DNA in the supernatant of DNase-digested PMA-stimulated neutrophils were defined as 100% NET-standard, and the plasma sample data were expressed as %NET-standard. The levels of MPO-DNA (%NET-standard) were significantly higher in the plasma from patients with COVID-19 (n = 16; 29.1% [IQR, 25.8, 41.5]) than in the plasma from healthy controls (n = 10; 13.4% [IQR, 12.4, 14.8]), as were the levels of NE-DNA (%NET-standard) (46.4% [IQR, 32.7, 53.7] vs. 12.1% [IQR, 9.9, 14.7], respectively, P &#60; 0.01; Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64644/64644fig01.jpg" /> 
Figure 1: Basic stages of a sandwich enzyme-linked immunosorbent assay method for measuring myeloperoxidase-DNA or neutrophil elastase-DNA in samples. (A) The wells are coated with an anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) capture antibody. (B) The remaining protein binding sites are blocked by blocking agents. (C) Samples containing NE-conjugated and MPO-conjugated DNA are added. (D) Limited DNase digestion is performed, and the sample is incubated in the wells to bind with the capture antibody. (E) A secondary peroxidase-labeled anti-DNA antibody is added. (F) Unbound secondary peroxidase-labeled anti-DNA antibody is removed. (G) The substrate 2,2&#39;-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is added, and the color development is monitored. Abbreviations: ABTS = 2,2&#39;-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); MPO = myeloperoxidase; NE = neutrophil elastase <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64644/64644fig01large.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/64644/64644fig02.jpg" /> 
Figure 2: Linearity of the relationships between the intensity values and the various dilutions of the neutrophil extracellular trap standard. The supernatant of DNase-digested phorbol 12-myristate 13-acetate-stimulated neutrophils was serially diluted, and the levels of myeloperoxidase-DNA and neutrophil elastase-DNA were assayed to generate a calibration curve. The absorbance units (AU) obtained from the undiluted neutrophil extracellular trap standard (NET-standard) were assigned as 100%NET, and plotting the intensity value at 405 nm against the %NET-standard revealed a linear relationship. Abbreviations: MPO = myeloperoxidase; NE = neutrophil elastase; NET = neutrophil extracellular trap <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64644/64644fig02large.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/64644/64644fig03.jpg" /> 
Figure 3: Dose-response of DNase I in neutrophil extracellular trap-DNA digestion. The sample was inserted into myeloperoxidase-coated wells. Then, DNase I (0 &#181;g/mL, 0.3 &#181;g/mL, 0.6 &#181;g/mL, or 0.9 &#181;g/mL) was added. After 15 min of digestion, the enzyme activity was stopped, and the remaining steps of the sandwich enzyme-linked immunosorbent assay were performed accordingly. The data are shown as the mean &#177; SD. N = 12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64644/64644fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64644/64644fig04v2.jpg" /> 
Figure 4: Plasma levels of myeloperoxidase-DNA and neutrophil elastase-DNA complexes in patients with COVID-19. The data are expressed as percentages of neutrophil extracellular trap standard (NET-standard) content and presented as medians and interquartile ranges. Patients with COVID-19, n = 16; healthy controls, n = 10. ** P &#60; 0.01 versus healthy controls. Abbreviations: HC = healthy controls; NET = neutrophil extracellular trap. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64644/64644fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Intra-assay coefficient of variability. Thirty samples were measured in duplicate to monitor individual coefficients of variability and, thus, determine the intra-assay coefficient of variability. Abbreviations: AU = absorbance units; CV = coefficients of variability <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64644/Table 1- R3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Inter-assay coefficient of variability. The samples were measured in quadruplicate on 10 different plates to monitor plate-to-plate variation and, thus, determine the inter-assay coefficient of variability. Abbreviations: AU = absorbance units; CV = coefficient of variability. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64644/Table 2- R3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Specificity test for capture antibodies. The plates were coated with iso-type control antibodies for anti-myeloperoxidase (MPO) and anti-neutrophil elastase (NE) to evaluate the specificity of the capture antibodies for the MPO-DNA and NE-DNA complexes. Abbreviations: AU = absorbance units; MPO = myeloperoxidase; NE = neutrophil elastase <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64644/Table 3- R3.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA at JoVE.com

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

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 ...

quantifying-myeloperoxidase-dna-neutrophil-elastase-dna-complexes

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.4K Views.  University of Science and Technology Chittagong (USTC). NET's hebben de laatste tijd de aandacht getrokken, maar het kwantificeren van NET's in vivo is een uitdaging gebleken. Dit protocol biedt een wenselijke, zeer gevoelige en waardevolle methode voor het onderzoeken van de kenmerken van NET's in klinische omgevingen. Deze techniek moet zich uitstrekken tot het evalueren van de ernst van sepsis of septische ARDS, acuut respiratoir distress syndroom.Het is algemeen aanvaard dat het reguleren van stortplaatsen of schade-geassocieerde molecu...

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

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 ...

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 ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Biology

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 ...

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. ...

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 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 ...

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 ...

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