Name: quantifying myeloperoxidase-dna and neutrophil elastase-dna complexes from neutrophil extracellular traps by using a modified sandwich elisa
Uploaded: 2023-05-12T12:25:00
Description: 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

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 o...

<ol>
	<li>Sil, P., Yoo, D. G., Floyd, M., Gingerich, A., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+measurement+of+extracellular+DNA+release+and+quantitative+NET+formation+in+human+neutrophils+in+vitro.">High throughput measurement of extracellular DNA release and quantitative NET formation in human neutrophils in vitro.</a> Journal of Visualized Experiments. (112), e52779 (2016).</li><li>Yoo, D. G., Floyd, M., Winn, M., Moskowitz, S. M., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NET+formation+induced+by+Pseudomonas+aeruginosa+cystic+fibrosis+isolates+measured+as+release+of+myeloperoxidase-DNA+and+neutrophil+elastase-DNA+complexes.">NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.</a> Immunology Letters. 160 (2), 186-194 (2014).</li><li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Papayannopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+immunity+and+disease.">Neutrophil extracellular traps in immunity and disease.</a> Nature Reviews Immunology. 18, 134-147 (2018).</li><li>Chamardani, T. M., Amiritavassoli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+NETosis+for+treatment+purposes:+Friend+or+foe.">Inhibition of NETosis for treatment purposes: Friend or foe.</a> Molecular and Cellular Biochemistry. 477 (3), 673-688 (2022).</li><li>Rao, A. N., Kazzaz, N. M., Knight, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+neutrophil+extracellular+traps+contribute+to+the+heightened+risk+of+thrombosis+in+inflammatory+diseases.">Do neutrophil extracellular traps contribute to the heightened risk of thrombosis in inflammatory diseases.</a> World Journal of Cardiology. 7 (12), 829-842 (2015).</li><li>S&#248;rensen, O. E., Borregaard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+-+The+dark+side+of+neutrophils.">Neutrophil extracellular traps - The dark side of neutrophils.</a> Journal of Clinical Investigation. 126 (5), 1612-1620 (2016).</li><li>Abrams, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+neutrophil+extracellular+traps+(NETs)+formation+independently+predicts+disseminated+intravascular+coagulation+and+mortality+in+critically+ill+patients.">A novel assay for neutrophil extracellular traps (NETs) formation independently predicts disseminated intravascular coagulation and mortality in critically ill patients.</a> American Journal of Respiratory and Critical Care Medicine. 200 (7), 869-880 (2019).</li><li>Brinkmann, V., Goosmann, C., K&#252;hn, L. I., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+quantification+of+in+vitro+NET+formation.">Automatic quantification of in vitro NET formation.</a> Frontiers in Immunology. 3, 413 (2012).</li><li>Zhao, W., Fogg, D. K., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+image-based+quantitative+method+for+the+characterization+of+NETosis.">A novel image-based quantitative method for the characterization of NETosis.</a> Journal of Immunological Methods. 423, 104-110 (2015).</li><li>Masuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NETosis+markers:+Quest+for+specific,+objective,+and+quantitative+markers.">NETosis markers: Quest for specific, objective, and quantitative markers.</a> Clinica Chimica Acta. 459, 89-93 (2016).</li><li>Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps.">Neutrophil extracellular traps.</a> Methods in Molecular Biology. 1982, 517-528 (2019).</li><li>Kano, H., Huq, M. A., Tsuda, M., Noguchi, H., Takeyama, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sandwich+ELISA+for+circulating+myeloperoxidase-+and+neutrophil+elastase-DNA+complexes+released+from+neutrophil+extracellular+traps.">Sandwich ELISA for circulating myeloperoxidase- and neutrophil elastase-DNA complexes released from neutrophil extracellular traps.</a> Advanced Techniques in Biology &amp; Medicine. 5 (1), 1000196 (2016).</li><li>Prevel, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+markers+of+neutrophil+extracellular+trap+are+linked+to+survival+but+not+to+pulmonary+embolism+in+COVID-19-related+ARDS+patients.">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><li>Schechter, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+extracellular+trap+(NET)+levels+in+human+plasma+are+associated+with+active+TB.">et al. extracellular trap (NET) levels in human plasma are associated with active TB.</a> PLoS One. 12, e0182587 (2017).</li><li>Papayannopoulos, V., Metzler, K. D., Hakkim, A., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+elastase+and+myeloperoxidase+regulate+the+formation+of+neutrophil+extracellular+traps.">Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.</a> Journal of Cell Biology. 191 (3), 677-691 (2010).</li><li>Gupta, S., Chan, W., Zaal, K. J., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+real-time+imaging+technique+to+quantify+NETosis+and+distinguish+mechanisms+of+cell+death+in+human+neutrophils.">A high-throughput real-time imaging technique to quantify NETosis and distinguish mechanisms of cell death in human neutrophils.</a> Journal of Immunology. 200 (2), 869-879 (2018).</li><li>Li, M., Lin, C., Leso, A., Nefedova, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+citrullinated+histone+H3+bound+DNA+for+detection+of+neutrophil+extracellular+traps.">Quantification of citrullinated histone H3 bound DNA for detection of neutrophil extracellular traps.</a> Cancers. 12 (11), 3424 (2020).</li><li>Th&#229;lin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+citrullinated+histones:+Development+of+an+improved+assay+to+reliably+quantify+nucleosomal+H3Cit+in+human+plasma.">Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.</a> Journal of Thrombosis and Haemostasis. 18 (10), 2732-2743 (2020).</li><li>Li, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAD4+is+essential+for+antibacterial+innate+immunity+mediated+by+neutrophil+extracellular+traps.">PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps.</a> Journal of Experimental Medicine. 207 (9), 1853-1862 (2010).</li><li>Zuo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+in+COVID-19.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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><li>Gong, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+potential+biomarkers+and+immune+features+of+sepsis+using+bioinformatics+analysis.">Identification of potential biomarkers and immune features of sepsis using bioinformatics analysis.</a> Mediators of Inflammation. 2020, 3432587 (2020).</li><li>Almansa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+correlates+of+organ+failure+extent+in+sepsis.">Transcriptomic correlates of organ failure extent in sepsis.</a> Journal of Infection. 70 (5), 445-456 (2015).</li></ol>

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 ...

NETs have attracted recent attention, but quantifying NETs in vivo has proven challenging. This protocol provides a desirable, highly sensitive, and valuable method for investigating the characteristics of NETs in clinical settings. This technique should extend to evaluate the severity of sepsis or septic ARDS, acute respiratory distress syndrome.It is widely accepted that regulating dumps or damage-associated molecular patterns is a key to improving the clinical condition of sepsis. The greatest advantage of this method includes the accurate quantification of circulating NET remnants like myeloperoxidase-DNA and neutrophil elastase-DNA in a short time. It is advisable to spin the samples before assay and avoid repeated thawing.It is important to follow the incubation times as given in the protocol. Begin by diluting freshly isolated polymorphonuclear neutrophils to 10, 000 cells per milliliter in phenol red-free RPMI 1640. Sieve them in 35-millimeter culture dishes.To induce neutrophil extracellular traps or NETs, first stimulate the polymorphonuclear neutrophils with 25 nanomolar PMA. Then partially digest the extracellular traps by adding 0.6 micrograms per milliliter of DNase I, and incubate the mixture for 50 minutes at room temperature. Now, add five-millimolar EDTA to stop the DNase activity, and collect the medium containing the synthesized NETs.Centrifuge to remove the cell debris. Collect the supernatants from four healthy controls. Mix store them at minus 80 degrees Celsius for further use.Pipette 100 microliters of diluted anti-myeloperoxidase containing 0.05 micrograms of the antibody into an ELISA plate. Now, cover the plate with an adhesive plastic cover to prevent sample evaporation, and incubate the sample overnight at four degrees Celsius to allow the binding of the capture antibodies. The next day, discard the diluted antibody solution from the wells, and pipette 300 microliters of wash solution into each well.Tap the plate dry on a paper towel to remove the excess PBS. Block each well of the plate with 200 microliters of the blocking buffer. Cover it with an adhesive plastic cover, and obstruct the wells by incubating it.After discarding the blocking solution from the wells and washing the plate three to four times, tap it dry on a paper towel. Next, pipette 25 microliters of plasma into all wells except the blank. And dilute it with 75 microliters of PBS, making the final well volume 100 microliters.Then add 100 microliters of PBS to the blank well. Mix the samples by placing the plate on a shaker for 10 seconds at 250 rpm at room temperature. Add two microliters of 100 full diluted DNase I to all wells, and place the sealed plate on the shaker for 10 seconds to thoroughly mix the samples.Incubate for 15 minutes at room temperature. Add one microliter of 0.5 molar EDTA to each well to stop the DNase reaction. Then place the sealed plate on the shaker for 15 seconds to thoroughly mix the samples.Finally, incubate the plate overnight at four degrees Celsius to allow the protein components of the NETs to attach to the capture antibodies. The next day, discard the solution from the wells. After multiple well washes, pipette 100 microliters of the diluted peroxidase-conjugated anti-DNA detection antibody into each well.Incubate the plate for 1 1/2 hours, and then discard the solution in the wells. After washing the wells thrice, pipette 100 microliters of ABTS substrate solution into each well, and incubate the sealed plate on a shaker in the dark. Stop the reaction by adding 50 microliters of two-molar sulfuric acid.Mix the well contents by carefully tapping the plate sides. Next, connect the microplate reader to the computer and launch the software application. From the status bar, create a new experiment and name it.Then set the plate reading parameters by selecting Absorbance as the read type, Endpoint as the read mode, and two as the wavelengths. Next, set lambda one as 405 nanometers, lambda two as 490 nanometers. Now, select the Off status for both Automix and Blanking while keeping the AutoCalibration as On.Then select Read entire plate under Strips.Finally, set the Column Wavelength Priority with Normal as the Carriage Speed, and then select Off under AutoRead. Place the plate well into the instrument drawer and close it. Click on Read so that the plate is read immediately.Read the absorbance of each well at 405 nanometers, and perform automatic subtraction of the assay medium absorbance from all unknown samples. Reliable standard calibration curves were obtained for both MPO-DNA and NE-DNA when the absorbance values did not exceed 0.93 and 0.9, respectively. The highest OD was obtained when 0.6 micrograms per milliliter of DNase I was applied.The inter-assay variability coefficients for the complexes in healthy controls were 1.871 and 0.987, respectively, while the COVID-19 patients showed variability coefficients of 2.532 and 2.010, respectively. The mean inter-assay variability coefficients for MPO-DNA and NE-DNA were 6.524 and 4.389, respectively. The capture antibodies'specificity to the MPO-DNA and NE-DNA complexes showed that the iso-type control antibodies reacted little to the complexes.The percentage of both complexes was higher in the plasma from patients with COVID-19 relative to the healthy controls. DNase digestion time should be optimized, otherwise excessive digestion will reduce the absorbance.

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

Research

JoVE Journal

Immunology and Infection

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 ...

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. ...

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 ...

Immunofluorescence Staining of Neutrophil Extracellular Traps in Tissue Samples

Begin by pipetting one drop of ready-to-use blocking solution with donkey serum onto the tissue samples. Then incubate the samples for 30 minutes at room temperature. Remove the excess blocking solution from the slides by tapping the edge against a hard surface.Dilute the primary antibodies in the antibody dilution buffer to prepare 100 microliters of the final solution for each sample. Set up the antibodies, one tube for each primary antibody and one for each isocontrol antibody. Evenly spread 100 microliters of isocontrol antibodies to one sample, and 100 microliters of myeloperoxidase and citrullinated histone H3, or myeloperoxidase and neutrophil elastase antibody dilution to the other sample.Place the slides in a wet chamber and store them overnight at four degrees Celsius. The following day, tap off excess primary antibody solution from the slides and stack them in a cuvette. Rinse the slides three times for five minutes each using Tris buffered saline with tween To remove the remaining primary antibody solution.Prepare two distinctly fluorescent secondary antibodies, donkey anti-goat for myeloperoxidase and donkey anti-rabbit for citrullinated histone H3 staining. Dilute each antibody to 7.5 micrograms per milliliter concentration in the antibody dilution buffer. Place the slides in a wet chamber and dispense 100 microliters of the secondary antibody solution onto each sample.Incubate them for 30 minutes at room temperature protected from light. Remove excess secondary antibody solution by tapping the slides. Put the slides in a slide rack.Submerge the slides three times for five minutes each in a staining jar filled with PBS to wash off unbound antibodies. Prepare the dappy solution and dilute it to a concentration of one microgram per milliliter with deionized water. Submerge the slide rack in a staining jar containing the dappy solution and incubate for five minutes at room temperature in the dark.Next, submerge the slide rack in a staining jar with PBS for five minutes to wash off any excess dappy solution. Finally, mount the samples using cover slips and mounting medium. The citrullinated histone H3 antibody only bound to the extracellular histones and showed no staining of intracellular histones.The human or mouse specific myeloperoxidase antibody showed no specific staining. While the universal human and mouse antibodies showed consistent good staining for myeloperoxidase. When no blocking agent was used, some mouse samples showed more non-specific and partial positive staining.When blocked, the five minutes blocking time showed good results compared to the 10 minutes blocking time. The higher temperatures in the microwave and water bath showed consistently moderate to good antigen retrieval. Whereas in a 60 degrees Celsius water bath there was only partially positive to no staining.For double staining, more favorable results were obtained for the samples incubated at 96 degrees Celsius water bath. However, exceeding the incubation time of 40 minutes at 96 degrees Celsius resulted in less intense antibody staining.