Special thanks to the personnel of the University of Georgia Health Center laboratory for their continuous support of our work on isolating human neutrophils. This work was supported by the start-up fund of Dr. Rada provided by UGA Office of Vice President for Research.

The Institutional Review Board of the University of Georgia approved the human subject study to collect peripheral blood from healthy volunteers (UGA# 2012-10769-06).5,7,8 Volunteers signed the required informed consent form before blood draw. The research performed in this article is in compliance with the ethical guidelines for medical research involving human subjects of the Declaration of Helsinki.
1. Isolation of Neutrophils from Peripheral Human Blood (Figure 1)
Note: There are several ways to isolate neutrophils from peripheral blood. The following protocol provides one possibility. This protocol yields large numbers of non-activated human neutrophils capable of releasing NETs upon external stimulation. Use 40 ml whole blood obtained from healthy volunteers by venipuncture.7,9
<ol>
	<li>Aliquot 20 ml blood into two 50 ml conical tubes. Add 10 ml 6% Dextran. Mix gently.</li>
	<li>After 20 min transfer the leukocyte-rich upper phase without taking any pelleted red blood cells into clean 50 ml conical tubes, and fill it up with sterile PBS.</li>
	<li>Centrifuge at 400 x g 10 min. Resuspend pellet in 4 ml sterile PBS.</li>
	<li>Prepare a 5-step Percoll gradient of 85%-80%-75%-70%-65% in two 15 ml conical tubes and layer 2 ml leukocyte suspension on top of each gradient.</li>
	<li>Centrifuge 800 x g for 30 min with brakes off.</li>
	<li>Collect all the cells (neutrophils) in the layers at the 75%/80% and 70%/75% interface.</li>
	<li>Wash cells twice in PBS (350 x g, 10 min, RT) and count cells using a hemocytometer.</li>
</ol>
2. Measurement of Kinetics of Extracellular DNA Release Using a Microplate Fluorimeter
Note: This method enables measurement of changes in fluorescence of a membrane-impermeable DNA-binding dye indicative of DNA release on a 96-well microplate format (Figure 2).
<ol>
	<li>Prepare a suspension of neutrophils in assay medium (HBSS + 1% (v/v) autologous serum + 5 mM glucose) at a concentration of 2 x 106 cells/ml.</li>
	<li>Add 5 &#181;mol/L of the membrane impermeable DNA-binding dye. Mix gently.</li>
	<li>Aliquot human neutrophils (50 &#181;l/well) onto 96-well black, transparent bottom microplates. Make sure the assay medium covers the entire bottom of the well. If not, tap the plate gently on the side.</li>
	<li>Warm up the plate containing neutrophils for 10 min at 37 &#176;C.</li>
	<li>In the meantime prepare solutions of stimuli at double of their final concentrations to be used (in 37 &#176;C warm assay medium). As a positive NET control use human neutrophils with 100 nM phorbol-myristate-acetate (PMA) for 4 hr to trigger maximal NET release.5,8 PMA stimulation for 4 hr ensures maximal NET release while spontaneous NET formation remains low5,8.</li>
	<li>Prepare the microplate fluorimeter for the measurement (4 hr, 37 &#176;C, 530 nm for excitation and 590 nm for emission).</li>
	<li>Stimulate neutrophils by adding 50 &#181;l stimulus solution to 50 &#181;l neutrophil suspensions. Use 0.5 &#181;g/ml saponin in one well to measure maximal DNA release signal.</li>
	<li>Place plate in reader and measure changes in fluorescence for 4 hr at every 2 min with no shaking.</li>
	<li>For data analysis, calculate normalized increases in fluorescence over time.
	<ol>
		<li>Subtract baseline fluorescence from endpoint values for all wells including saponin (Figure 2A, B). Rise in saponin fluorescence should be the highest signal. It is referred to as &#34;maximal DNA release&#34; (Figure 2A).</li>
		<li>Calculate percentages of DNA release in unknown samples by dividing increases in fluorescence of unknown samples by maximal DNA release.</li>
		<li>Average results of replicates and present them as &#34;% of maximal DNA release&#34; (Figure 2C).</li>
	</ol>
	</li>
</ol>
3. Quantitation of NET Formation by MPO-DNA and HNE-DNA ELISA Assays
Note: These assays modified (MPO-DNA) or established (HNE-DNA) in our laboratory quantitate NET formation by measuring levels of MPO-DNA and HNE-DNA complexes.5
<ol>
	<li>Coat 96-well high-binding capacity ELISA plates overnight with capture antibodies (50 &#956;l/well): anti-MPO (1:2,000 dilution) or anti-HNE (1:2,000).</li>
	<li>Wash ELISA plates three times with PBS (200 &#956;l/well) and block them with 5% BSA and 0.1% human serum albumin for 2 hr at RT (200 &#956;l/well). Wash three times with PBS.</li>
	<li>Seed neutrophils on 96-well microplates at a density of 100,000 cells/well in 100 &#181;l assay medium, and stimulate NET formation. Incubate cells: 4 hr for 37 &#176;C.</li>
	<li>Perform a limited DNase digestion by adding 2 U/ml DNase to neutrophil supernatants, mix them well. Keep them at RT.</li>
	<li>Stop the DNase after 15 min by adding 11 &#181;l 25 mM EGTA (final concentration 2.5 mM). Mix well. Collect supernatants in clean microfuge tubes. Spin samples to get rid of cell debris at 300 x g for 5 min at RT. Transfer their supernatants into clean microfuge tubes. Keep them on ice until ready.</li>
	<li>Use an aliquot of a previously prepared &#34;NET standard&#34;. 
	Note: The NET-standard is a mix of DNase-digested supernatants of PMA-stimulated neutrophils obtained from at least 5 different, independent human donors.
	<ol>
		<li>To use the same standard for a long time, collect and aliquot a large volume of neutrophil supernatants from each individual donor. Collecting for example 2 ml of DNase-digested supernatants of PMA-stimulated neutrophils will result in 200 aliquots (10 &#181;l each) enough for 200 ELISA plates. Data characterizing the NET standard are found in Figure 5.</li>
		<li>Stimulate human neutrophils with 100 nM PMA for 4 hr and apply DNase digest to their supernatants according steps 3.4-3.5.</li>
		<li>Collect, pool, aliquot (10 &#181;l) and freeze (-20 &#176;C) neutrophil supernatants.</li>
		<li>To prepare the &#34;NET-standard&#34;, thaw one aliquot/donor obtained from at least five separate donors, mix them and keep them on ice.</li>
		<li>Discard thawed samples and use fresh ones for the next experiment.</li>
		<li>Prepare a 1:2 serial dilution of the NET-standard in PBS+EGTA.</li>
	</ol>
	</li>
	<li>Dilute DNase1-digested samples (standard and unknown supernatants) 20-fold in PBS+EGTA and aliquot them on ELISA plates coated with capture antibodies.</li>
	<li>Incubate ELISA plates over night at 4 &#176;C and wash them three times with PBS.</li>
	<li>Apply detection antibody solution (100 &#181;l/well): horse radish peroxidase conjugated anti-DNA antibody (1:500, mouse). Incubate for 1 hr in dark.</li>
	<li>After four washes with PBS add TMB peroxidase substrate: 100 &#181;l/well, 30 min. Blue coloration is an indication for peroxidase activity (NETs present).</li>
	<li>Stop reaction by adding 100 &#956;l/well 1 M HCl. The blue solutions will turn yellow (Figure 5A).</li>
	<li>Read absorbance at 450 nm using a microplate photometer.</li>
	<li>For data analysis, calculate the amount of MPO-DNA or HNE-DNA complexes compared to the NET standard.
	<ol>
		<li>Subtract the background optical density value of the assay medium from all samples.</li>
		<li>Plot the absorbance values (X-axis) of the diluted standard samples against their relative NET content (Y-axis) (Figure 5A).</li>
		<li>Using the nonsaturated range of this standard curve establish a trend line (use best exponential fit from the software used) (Figure 5A). The equation of this trend line determines the conversion of OD values to quantitative amounts of NETs.</li>
		<li>Insert the measured OD values of the unknowns into the trend-line equation from 3.13.3 that will give the amount of NETs in the unknown sample as percentage of the NET-standard (Figure 5A).</li>
		<li>Calculate averages of replicates (triplicates) and present the final data as &#34;amount of MPO-DNA or HNE-DNA complexes (% of standard)&#34; (Figure 5C).</li>
	</ol>
	</li>
</ol>

<ol><li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+kill+bacteria%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neutrophil extracellular traps kill bacteria.</a> Science. 303, 1532-1535 (2004).</li>
<li>Fuchs, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+cell+death+program+leads+to+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Novel cell death program leads to neutrophil extracellular traps.</a> J Cell Biol. 176, 231-241 (2007).</li>
<li>Kessenbrock, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Netting+neutrophils+in+autoimmune+small-vessel+vasculitis%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Netting neutrophils in autoimmune small-vessel vasculitis.</a> Nat Med. 15, 623-625 (2009).</li>
<li>Leffler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Degradation+of+neutrophil+extracellular+traps+co-varies+with+disease+activity+in+patients+with+systemic+lupus+erythematosus%5BTitle%5D)+AND+%22Arthritis+Res+Ther%22%5BJournal%5D)">Degradation of neutrophil extracellular traps co-varies with disease activity in patients with systemic lupus erythematosus.</a> Arthritis Res Ther. 15, R34(2013).</li>
<li>Yoo, D. G., Floyd, M., Winn, M., Moskowitz, S. M., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NET+formation+induced+by+Pseudomonas+aeruginosa+cystic+fibrosis+isolates+measured+as+release+of+myeloperoxidase-DNA+and+neutrophil+elastase-DNA+complexes%5BTitle%5D)+AND+%22Immunol+Lett%22%5BJournal%5D)">NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.</a> Immunol Lett. 160, 186-194 (2014).</li>
<li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+hypercitrullination+mediates+chromatin+decondensation+and+neutrophil+extracellular+trap+formation%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.</a> J Cell Biol. 184, 205-213 (2009).</li>
<li>Pang, L., Hayes, C. P., Buac, K., Yoo, D. G., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudogout-associated+inflammatory+calcium+pyrophosphate+dihydrate+microcrystals+induce+formation+of+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps.</a> J Immunol. 190, 6488-6500 (2013).</li>
<li>Yoo, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Release+of+cystic+fibrosis+airway+inflammatory+markers+from+Pseudomonas+aeruginosa-stimulated+human+neutrophils+involves+NADPH+oxidase-dependent+extracellular+DNA+trap+formation%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation.</a> J. Immunol. 192, 4728-4738 (2014).</li>
<li>Brinkmann, V., Laube, B., Abu Abed,, Goosmann, U., Zychlinsky, C., A, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps%2C+how+to+generate+and+visualize+them%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Neutrophil extracellular traps, how to generate and visualize them.</a> J. Vis. Exp. , (2010).</li>
<li>Rada, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pyocyanin-enhanced+neutrophil+extracellular+trap+formation+requires+the+NADPH+oxidase%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase.</a> PLoS One. 8, e54205(2013).</li>
<li>Neumann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+antimicrobial+peptide+LL-37+facilitates+the+formation+of+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps.</a> Biochem J. 464, 3-11 (2014).</li>
<li>Leon, S. A., Shapiro, B., Sklaroff, D. M., Yaros, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Free+DNA+in+the+serum+of+cancer+patients+and+the+effect+of+therapy%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Free DNA in the serum of cancer patients and the effect of therapy.</a> Cancer Res. 37, 646-650 (1977).</li>
<li>Raptis, L., Menard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitation+and+characterization+of+plasma+DNA+in+normals+and+patients+with+systemic+lupus+erythematosus%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus.</a> J Clin Invest. 66, 1391-1399 (1980).</li>
<li>Chang, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elevated+cell-free+serum+DNA+detected+in+patients+with+myocardial+infarction%5BTitle%5D)+AND+%22Clin+Chim+Acta%22%5BJournal%5D)">Elevated cell-free serum DNA detected in patients with myocardial infarction.</a> Clin Chim Acta. 327, 95-101 (2003).</li>
</ol>

The authors have nothing to disclose.

NETs represent a fascinating novel mechanism by which neutrophils kill pathogens.1 Although the literature of NETs has been continuously expanding over the last ten years since their discovery, several important questions related to their role in biology, mechanism and regulation remain unclear. Appropriate methodology has to be developed to measure NETs, this very unique antimicrobial mechanism. This article describes methods that can be used to quantitate NETs in a high throughput manner. The first assay fol...

NET formation is a novel mechanism by which neutrophils fight pathogens.1 The core of NETs is nuclear DNA.1 This DNA network is associated with neutrophil granule proteins and histones.1 The main form of NET formation requires the death of neutrophils characterized by chromatin decondensation, disappearance of granular and nuclear membranes, translocation of neutrophils elastase to the nucleus, citrullination of histones and finally the spill of DNA-based NETs.2 NETs entrap and kill a wide variety of microbes and are an essential part of the innate immune weapon repertoire. Uncontrolled NET formation has, however, been linked to numerous autoinflammatory diseases.3,4 Despite their increasingly established relevance, little is known about the mechanism and regulation of NET release. 
Neutrophils dying by releasing NETs are different from apoptotic or necrotic neutrophils.3,5 NET-releasing neutrophils show several features that are characteristic for NET formation. Granule components are associated with DNA in NETs.1 Myeloperoxidase (MPO) and human neutrophil elastase (HNE) are both found in primary granules in resting cells but are translocated to the nucleus to bind to DNA in NETs.1 MPO-DNA and HNE-DNA complexes are specific for NETs, do not occur in apoptotic or necrotic neutrophils.1,3,5 Chromatin decondensation is another feature typical for NETosis.2 NET release also requires citrullination of histones by peptidyl aminidase 4 (PAD4).6 Citrullinated histones are hallmarks of neutrophils that underwent NET release.6
Here three methods are introduced that in combination provide excellent tools to quantitate NETs on a high-throughput scale. The first assay has been used on the field with different changes and quantitates extracellular DNA release in a microplate format. The second and third assays provide confirmation of NETs by measuring NET-specific MPO-DNA and HNE-DNA complexes.

<table><tbody><tr><td>Anti-Human Neutrophil Elastase&amp;nbsp; Rabbit Ab&amp;nbsp;</td><td>Calbiochem</td><td>481001</td><td>1:2,000x coated</td></tr><tr><td>Anti-Myeloperoxidase Ab (Rabbit)</td><td>Millipore</td><td>07-496</td><td>1:2,000x coated</td></tr><tr><td>DNase-1</td><td>Roche</td><td>10-104-159-001</td><td>1 &amp;#181;g/ml used for digestion</td></tr><tr><td>20 mM EGTA/ PBS</td><td>Sigma-Aldrich</td><td>E3889-25G</td><td></td></tr><tr><td>2.5 mM EGTA/PBS</td><td>Sigma-Aldrich</td><td>E3889-25G</td><td></td></tr><tr><td>Cell death detection ELISA Anti-DNA POD</td><td>Roche</td><td>11544675001</td><td>1:500x&amp;nbsp;</td></tr><tr><td>Eon Microplate Spectrophotometer</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 All-in-One microplate software</td><td>Biotek</td><td></td><td>analytical tool (ELISA)</td></tr><tr><td>Sytox orange</td><td>Life Technology</td><td>S11368</td><td>0.2% final concentration/volume</td></tr><tr><td>1 M Hepes</td><td>Cellgro</td><td>25-060-Cl</td><td>Use 10 mM final concentration.</td></tr><tr><td>1 M glucose</td><td>Sigma</td><td></td><td>Use 5 mM final concentration.</td></tr><tr><td>HBSS</td><td>Corning</td><td>21-023-CM</td><td></td></tr><tr><td>Varioskan Flash Ver.2.4.3</td><td>Thermoscientific</td><td></td><td></td></tr><tr><td>PMA</td><td>Sigma</td><td>P 8139</td><td>100 nM final used</td></tr><tr><td>ELISA Plate</td><td>Greiner bio-one</td><td>655061</td><td></td></tr><tr><td>Conical tubes 15 ml</td><td>Thermoscientific</td><td>339650</td><td></td></tr><tr><td>Conical tubes 50 ml</td><td>Thermoscientific</td><td>339652</td><td></td></tr><tr><td>Percoll (pH 8.5-9.5)&amp;nbsp;</td><td>Sigma</td><td>P 1644</td><td>Sodium Chloride, Sigma, S7653-250G</td></tr><tr><td>Dextran</td><td>Spectrum</td><td>D1004</td><td></td></tr><tr><td>RPMI 1640 media</td><td>Corning Cellgro</td><td>17-105-CV</td><td></td></tr><tr><td>96 well assay plate black plate clear bottom</td><td>Costar</td><td>3603</td><td></td></tr></tbody></table>

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.

The figures in this manuscript describe the method of neutrophil isolation, experimental procedures, and present representative results with explanation of data analysis. Figure 1 shows the sequential steps of human neutrophil preparation. This protocol represents only one possible way of neutrophil isolation. It yields large amounts of resting neutrophils capable of releasing NETs upon stimulation. Figure 2 shows how the fluorescence-based DNA release as...

Watch this Scientific Journal Video about High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro at JoVE.com

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.

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 ...

high-throughput-measurement-extracellular-dna-release-quantitative

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13.1K Views.  University of Georgia. High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

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

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

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 ...

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

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

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

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

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

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

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

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

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

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. ...

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

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

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

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

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 ...

Biology

An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

Source: Najmeh, S., et al. <a href="https://app.jove.com/v/52687">Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro technique to generate and isolate neutrophil extracellular traps (NETs). Neutrophils are isolated from peripheral blood and stimulated with phorbol myristate acetate (PMA) to produce NETs. During incubation, the neutrophils undergo a process of cell death (NETosis) to release NETs comprising decondensed chromatin mixed with granular cytoplasmic proteins.

Source: Najmeh, S., et al. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. J. Vis. Exp. (2015)
This video demonstrates an ...

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

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