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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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 sp...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Step Polymorphs</td>
			<td>Accurate Chemical and Scientific Corporation</td>
			<td>AN221725</td>
			<td>Isolation of PMN&#39;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.&#160;</td>
		</tr>
		<tr>
			<td>ABTS tablets</td>
			<td>Sigma-Aldrich Merck</td>
			<td>11 204 521 001&#160;</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>&#160;07-496-I</td>
			<td>Store at 2-8 &#176;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 &#176;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&#8211;8 &#176;C. stable for 2 year.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>New England BioLabs</td>
			<td>M0303M</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>IgG, rabbit, Isotype Control</td>
			<td>GENETEX, Inc.</td>
			<td>GTX35035</td>
			<td>Store as concentrated solution at 2&#8211;8 &#176;C.</td>
		</tr>
		<tr>
			<td>IgG1, mouse Isotype Control, clone Ci4</td>
			<td>Merck&#160;</td>
			<td>MABC002</td>
			<td>Store as concentrated solution at 2&#8211;8 &#176;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&#160;</td>
			<td>Any microplate plate reader capable of reading wavelengths from 405&#8211;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>&#160;bottle 2. Store at 2&#8211;8 &#176;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&#39;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>&#160;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 sam...

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

Research

JoVE Journal

Immunology and Infection

3.1K Views.  University of Science and Technology Chittagong (USTC). 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.

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

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 ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

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

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

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

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

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 ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Staining and Imaging to Visualize Neutrophil Extracellular Traps

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 &#181;m thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.
Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

Author Spotlight: Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues 

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...

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.

Methods Article

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