The authors gratefully acknowledge Drs James Roth, William Nauseef and Kayoko Kimura and Mr Tom Skadow for assistance with development of the canine neutrophil protocols. RDG is supported by a Wellcome Trust Fellowship ref: WT093767MA.

All experiments were performed with ethical permission from the Iowa State University Institutional Animal Care and Use Committee.
1. Blood Collection
<ol>
	<li>Draw 9 ml of blood from a saphenous, cephalic or jugular vein directly into anticoagulant (e.g., ethylenediaminetetraacetic acid (EDTA), 1.8 mg K2EDTA/ml blood) vacutainers, or into a syringe with immediate transfer to EDTA-containing blood tubes. Gently roll or invert the blood tubes to ensure adequate mixing of the blood and anti-coagulant.</li>
</ol>
2. Neutrophil Isolation
<ol>
	<li>In a 50 ml conical tube, mix blood with an equal volume of sterile room temperature 3% dextran-500 (made up in 0.9% sterile saline) by gentle inversion. Leave standing upright for 18-20 min at room temperature. Transfer the straw colored upper layer to a fresh tube until it can no longer be collected without contamination by the lower red layer. The erythrocytes in the original tube can be discarded.</li>
	<li>Centrifuge the supernatant at 500 x g for 10 min at 4 &#176;C. Draw off and discard the supernatant. Manually re-suspend the cell pellet in 10 ml room temperature sterile phosphate buffered saline (PBS). Note that vortexing should not be used to re-suspend neutrophils at any stage of this protocol. Manually drawing off, rather than pouring off supernatants is also recommended throughout the protocol to maximize cell yield.</li>
	<li>Add 10 ml of a room temperature polysucrose and sodium diatrizoate cell separation solution (e.g., Histopaque 1077) to a 50 ml conical tube. Carefully layer the cell-containing solution over the cell separation solution. Note that using cell separation solution which has not equilibrated to room temperature may reduce cell yield.</li>
	<li>Centrifuge at 300 x g for 30 min at room temperature with the brake off.</li>
	<li>As neutrophils are in the lowest layer of the gradient, draw off and discard the upper 3 layers, composed of an upper layer of plasma and platelets, a narrow white band of mononuclear cells and a clear layer of the density gradient medium.</li>
	<li>To lyse any remaining erythrocytes, re-suspend the neutrophil pellet in 10 ml of room temperature water.</li>
	<li>After 30 sec, restore tonicity by adding 10 ml of sterile room temperature 1.8% sodium chloride and mix by gentle inversion.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C. Draw off the supernatant and discard.</li>
	<li>If the pellet is still red, repeat the lysis step. If the pellet is white (or if lysis has already been performed twice), gently re-suspend cells in 10 ml of sterile PBS.</li>
	<li>Count cells using an automated counter or a hemocytometer. Typical yields are at least 106 cells per ml of blood.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C. Draw off the supernatant and discard.</li>
	<li>Re-suspend cells to the desired concentration in sterile room temperature PBS. For the DNA release assay described in section 2, resuspend at 5 x 106 cells per ml.</li>
	<li>If desired, transfer a small volume of cells directly or using a cytocentrifuge to a glass slide. Allow cells to dry and stain using a rapid fixation and staining kit according to the manufactures&#39; instructions. If the neutrophil isolation has been successful, when examined under the microscope at least 95% of nucleated cells should be granulocytes (neutrophils, basophils, eosinophils) with minimal erythrocyte contamination.</li>
</ol>
3. DNA Release Assay
<ol>
	<li>Set up the DNA release assay immediately after neutrophil isolation.</li>
	<li>If cell clumping is present on examination of the stock solution by light microscopy, pass the cell suspension through a sterile 70 &#181;m sterile filter immediately before setting up the assay.</li>
	<li>To each test well of a sterile 96 well plate, add 5 x 104 cells/well; agonist and Roswell Park Memorial Institute-1640 (RPMI) medium without phenol red and supplemented with 0.5% heat-inactivated fetal calf serum to a final volume of 100-200 &#181;l. Include at least two blank wells per agonist in which the neutrophil stock solution is replaced by an equal volume of PBS. Note that setting up blank wells for each agonist is important in ruling out DNA contamination of the agonist or interference by the agonist with the fluorescence assay.</li>
	<li>Incubate at 39 &#176;C for 30 min in a carbon dioxide (4%) incubator.</li>
	<li>Add agonists at desired concentrations and a cell impermeable nucleic acid dye. Note the optimal concentration of cell impermeable dye will vary between different nucleic acid dyes. Non-stimulated controls in which agonists are replaced by an equal volume of media should be included in each experiment. 
	NOTE: It is desirable to dilute agonists in similar volumes of culture medium to maintain consistent conditions between wells. Final well volumes of 200 &#181;l have been used successfully and if this is altered, well volumes should remain identical for all conditions. Phorbol-12-myristate-13-acetate (PMA) (final concentration &#8805;0.1 &#181;M) or platelet activating factor (PAF) (final concentration &#8805;31 &#181;M) can be used as positive controls. Agonists should be measured at least in duplicate.</li>
	<li>Incubate at 39 &#176;C. Measure fluorescence using a microplate reader after 2 hours or at desired intervals. Note that optimal time intervals will vary with agonists used. 
	NOTE: Wavelength will depend on dye employed.</li>
	<li>Subtract blank fluorescence before calculating mean fluorescence.</li>
	<li>To allow comparison between individuals and between plates, calculate a fold change in fluorescence by dividing the mean fluorescence of stimulated wells by the mean fluorescence of non-stimulated wells.</li>
</ol>

<ol><li>Schiffman, J. D., Breen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+oncology%3A+what+dogs+and+other+species+can+teach+us+about+humans+with+cancer%5BTitle%5D)+AND+%22Philos+Trans+Rl+Soc+B+Biol+Sci%22%5BJournal%5D)">Comparative oncology: what dogs and other species can teach us about humans with cancer.</a> Philos Trans Rl Soc B Biol Sci. 370, 1673(2015).</li>
<li>Williams, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+of+canine+leucocyte+antigens%3A+a+significant+advance+in+canine+immunology%5BTitle%5D)+AND+%22Vet+J%22%5BJournal%5D)">Studies of canine leucocyte antigens: a significant advance in canine immunology.</a> Vet J. 153 (1), 31-39 (1997).</li>
<li>Eckhart, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Duplication+of+the+caspase-12+prodomain+and+inactivation+of+NLRC4%2FIPAF+in+the+dog%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Duplication of the caspase-12 prodomain and inactivation of NLRC4/IPAF in the dog.</a> Biochem Biophys Res Commun. 384 (2), 226-230 (2009).</li>
<li>Eckhart, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+novel+mammalian+caspases+reveals+an+important+role+of+gene+loss+in+shaping+the+human+caspase+repertoire%5BTitle%5D)+AND+%22Mol+Biol+Evol%22%5BJournal%5D)">Identification of novel mammalian caspases reveals an important role of gene loss in shaping the human caspase repertoire.</a> Mol Biol Evol. 25 (5), 831-841 (2008).</li>
<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 (5663), 1532-1535 (2004).</li>
<li>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps%3A+is+immunity+the+second+function+of+chromatin%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Neutrophil extracellular traps: is immunity the second function of chromatin.</a> J Cell Biol. 198 (5), 773-783 (2012).</li>
<li>Chuammitri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chicken+heterophil+extracellular+traps+%28HETs%29%3A+novel+defense+mechanism+of+chicken+heterophils%5BTitle%5D)+AND+%22Vet+Immunol+Immunopathol%22%5BJournal%5D)">Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils.</a> Vet Immunol Immunopathol. 129 (1-2), 126-131 (2009).</li>
<li>Palić, D., Ostojić, J., Andreasen, C. B., Roth, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fish+cast+NETs%3A+neutrophil+extracellular+traps+are+released+from+fish+neutrophils%5BTitle%5D)+AND+%22Dev+Comp+Immunol%22%5BJournal%5D)">Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils.</a> Dev Comp Immunol. 31 (8), 805-816 (2007).</li>
<li>Poirier, A. C., Schmitt, P., Rosa, R. D., Vanhove, A. S., Kieffer-Jaquinod, S., Rubio, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+histones+and+DNA+traps+in+invertebrate+immunity%3A+evidences+in+Crassostrea+gigas%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas.</a> J Biol Chem. 289 (36), 24821-24831 (2014).</li>
<li>Robb, C. T., Dyrynda, E. A., Gray, R. D., Rossi, A. G., Smith, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invertebrate+extracellular+phagocyte+traps+show+that+chromatin+is+an+ancient+defense+weapon%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Invertebrate extracellular phagocyte traps show that chromatin is an ancient defense weapon.</a> Nat Commun. 5, 4627(2014).</li>
<li>Kaplan, M. J., Radic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps%3A+double-edged+swords+of+innate+immunity%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Neutrophil extracellular traps: double-edged swords of innate immunity.</a> J Immunol. 189 (6), 2689-2695 (2012).</li>
<li>Menegazzi, R., Decleva, E., Dri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Killing+by+neutrophil+extracellular+traps%3A+fact+or+folklore%3F%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Killing by neutrophil extracellular traps: fact or folklore?</a> Blood. 119 (5), 1214-1216 (2012).</li>
<li>Parker, H., Albrett, A. M., Kettle, A. J., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myeloperoxidase+associated+with+neutrophil+extracellular+traps+is+active+and+mediates+bacterial+killing+in+the+presence+of+hydrogen+peroxide%5BTitle%5D)+AND+%22J+Leukoc+Biol%22%5BJournal%5D)">Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide.</a> J Leukoc Biol. 91 (3), 369-376 (2012).</li>
<li>Martinod, K., Wagner, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thrombosis%3A+tangled+up+in+NETs%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Thrombosis: tangled up in NETs.</a> Blood. 123 (18), 2768-2776 (2014).</li>
<li>Pratesi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibodies+from+patients+with+rheumatoid+arthritis+target+citrullinated+histone+4+contained+in+neutrophils+extracellular+traps%5BTitle%5D)+AND+%22Ann+Rheum+Dis%22%5BJournal%5D)">Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps.</a> Ann Rheum Dis. 73 (7), 1414-1422 (2014).</li>
<li>Nauseef, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+human+neutrophils+from+venous+blood%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Isolation of human neutrophils from venous blood.</a> Methods Mol Biol. 1124, 13-18 (2014).</li>
<li>Gray, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+conventional+protein+kinase+C+%28PKC%29+is+critical+in+the+generation+of+human+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22J+Inflamm+%28Lond%29%22%5BJournal%5D)">Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps.</a> J Inflamm (Lond). 10 (1), 12(2013).</li>
<li>Jeffery, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dogs+cast+NETs+too%3A+Canine+neutrophil+extracellular+traps+in+health+and+immune-mediated+hemolytic+anemia%5BTitle%5D)+AND+%22Vet+Immunol+Immunopathol%22%5BJournal%5D)">Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia.</a> Vet Immunol Immunopathol. 168 (3-4), 262-268 (2015).</li>
<li>McCracken, J. M., Allen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+human+neutrophil+apoptosis+and+lifespan+in+health+and+disease%5BTitle%5D)+AND+%22J+Cell+Death%22%5BJournal%5D)">Regulation of human neutrophil apoptosis and lifespan in health and disease.</a> J Cell Death. 7, 15-23 (2014).</li>
</ol>

The authors have nothing to disclose.

The DNA release assay presented is a readily quantifiable assay for extracellular DNA. The method was adapted from similar techniques used to assess NET formation in other species, but centrifugation speeds, agonist concentrations and incubation times have been altered to optimize the method for use with canine neutrophils8,17,18. Similar adjustments could be made to adapt the method for other species. The assay is straightforward and inexpensive when compared with other methods for measuring NETosis such as flow cytometry or image analysis. The method also does not require antibodies against NET components, so is ideal for use in dogs for whom commercially produced, validated species-reactive antibodies are often unavailable.
The major limitation of the method is the lack of specificity of cell-impermeable nucleic acid dyes for NET formation. These dyes also enter dead and dying cells, bind DNA and fluoresce. Therefore, fluorescence is not specific for NET formation. Use of other techniques (e.g., immunofluorescence) in parallel with the quantitative assay to visually confirm agonists are inducing NET formation rather than other forms of cell death is strongly recommended. The assay can also potentially be used as a general screening assay for cell death, for example as a means of quantifying cytotoxicity ex vivo.
The protocol is simple, but there are critical steps necessary to ensure success. Firstly, as noted in the protocol, to avoid excessive cell loss during neutrophil isolation it is important that supernatants are drawn off with a pipette rather than discarded by inverting tubes. Secondly, success of the assay relies on the non-stimulated control cells remaining in a viable and resting state throughout the incubation. Neutrophils can become activated at any stage during the protocol so careful attention should be paid to atraumatic blood collection technique and avoiding traumatic handling (e.g., vortexing) during neutrophil isolation. It is also important to remember that neutrophils have a limited lifespan so delay in isolating cells or setting up the assay should be avoided19. If prolonged incubation with agonists is attempted, viability of the non-stimulated cells over this time period should be confirmed.
As shown in Figure 1, there is considerable variation between dogs in their response to NET agonists. However, the assay should be reasonably consistent between replicates from the same animal, with an intra-assay variation of 9% based on 10 duplicates18. If there are large variations between replicates, cell clumping is likely responsible. Measures to reduce cell clumping include maintaining cells in PBS rather than a calcium containing buffer prior to setting up the DNA assay, treating cells gently throughout isolation, and filtering the cell stock suspension prior to plating cells.
If these steps are followed, this protocol allows quantification of NET formation in response to a wide range of experimental conditions and agonists. Such studies can deepen our understanding of the role of NETs in canine health and disease. For example, this technique allows comparison of NET release between healthy dogs and those with immunosuppressive or autoimmune diseases in which NETosis may be impaired or enhanced. Alternatively, when combined with other techniques such as immunofluorescence, the assay can be used to identify novel agonists capable of inducing NETosis and novel inhibitors of NETosis.

There are over 70 million pet dogs in the USA alone1. As valued family members, these animals often receive cutting edge medical care. Equally because they share our environment, dogs can provide insights into the pathogenesis and treatment of human disease1. However, whether translating discoveries in human medicine into veterinary treatments or vice versa, it is important to thoroughly characterize species variations even in highly conserved systems such as the innate immune response. Examples of differences between the canine and human innate immune system include high expression CD4 on dog neutrophils2; the absence of a functional homolog of the cytoplasmic flagellin sensor IPAF in dogs3 and the expression of a caspase 1/4 hybrid in carnivores4.
Neutrophil extracellular traps (NETs) are a relatively recently discovered component of innate immunity5. NETs are networks of DNA, nuclear and granular proteins released in response to a wide range of inflammatory or infectious stimuli6. NET-like structures have been demonstrated across many species including chickens7, fish8, mollusks9 and acoelomates10, but there are species variations. For example, murine neutrophils respond more slowly to NETosis stimuli than human neutrophils, and form less diffuse NETs11. There is a large body of evidence from multiple species that NETs entrap microbes, and more controversially may be directly involved in killing pathogens12,13. However, NET components also enhance tissue damage, promote thrombosis and act as autoantigens14,15. The balance between the beneficial and deleterious effects of NETs may vary between different diseases and different species, suggesting it is important to investigate NETs both in the species and condition of interest.
Here we describe a simple protocol for inducing and measuring the release of NETs by canine neutrophils. This method is similar to those used to isolate neutrophils16 and induce NETosis in other species, but conditions such as agonist concentration and incubation time have been optimized for canine neutrophils. A similar NET quantification DNA release assay has also been described in other species but the method presented here is also optimized for dogs8,17,18.

<table><tbody><tr><td>Plastic Whole Blood tube with spray-coated K&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>BD</td><td>367835</td><td></td></tr><tr><td>Histopaque-1077</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>Dextran-500</td><td>Accurate chemical and scientific corp.</td><td>AN228410</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>ThermoFisher scientific</td><td>20012043</td><td></td></tr><tr><td>Fetal bovine calf serum, heat inactivated</td><td>ThermoFisher scientific</td><td>10100139</td><td></td></tr><tr><td>RPMI Media 1640, without phenol red or L-glutamine</td><td>ThermoFisher scientific</td><td>32404-014</td><td>Should be free from phenol red</td></tr><tr><td>96 well flat bottomed sterile polystyrene plate</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich</td><td>P1585</td><td></td></tr><tr><td>Platelet activating factor</td><td>Sigma-Aldrich</td><td>P4904</td><td></td></tr><tr><td>SYTOX Green Nucleic Acid Stain</td><td>ThermoFisher scientific</td><td>S7020</td><td></td></tr><tr><td>Synergy 2 Multi-Mode Reader</td><td>BioTek</td><td>NA</td><td></td></tr></tbody></table>

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.

Using this protocol, there should be a strong fold change in fluorescence after stimulating neutrophils with the positive controls, PMA and PAF. As illustrated in Figure 1B, stimulation of canine neutrophils with 31 &#181;M PAF for 1 hr results in a mean 4.0-fold increase in fluorescence compared with non-stimulated cells (range 2.0-5.8, n = 5 dogs)18. PMA at 0.1 &#181;M (Figure 1A) is a slower agonist which does not result in an increase in fluorescence until 2 hr but ultimately produces a similar fold increase in fluorescence (mean: 3.3, range 1.36-5.4, n = 5 dogs)18. Despite its slower kinetics, there are advantages to PMA as a positive control: it is relatively inexpensive compared with high concentrations of PAF and it has been widely used in murine and human studies.
Fluorescence of nucleic acid dyes is not specific for NET formation as it will also occur if there is cell death by other mechanisms or if reagents are contaminated with DNA. As shown in Figure 2, a commercial LPS preparation produced very high fluorescence in the absence of neutrophils, presumably due to contamination with bacterial DNA. Confirming NET formation through other methods such as immunofluorescence is recommended.
<img alt="Figure 1" src="/files/ftp_upload/54726/54726fig1.jpg" /> 
Figure 1: PMA and PAF increase fluorescence of a cell impermeable nucleic acid dye with different kinetics. Neutrophils were stimulated with 0.1 &#181;M PMA (A) or 31 &#181;M PAF (B) in the presence of the cell impermeable nucleic acid dye. Fluorescence was measured at hourly intervals and the fold change in fluorescence of stimulated cells compared to non-stimulated cells calculated. Fluorescence increased significantly between 1 and 2 hr for PMA (repeated measures one-way ANOVA followed by multiple t-tests with Bonferroni&#39;s correction, multiplicity adjusted p = 0.04; error bars are mean &#177; standard error for 5 individuals) but had already plateaued for PAF by 1 hr. This figure has been modified from Jeffery U, et al. Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia. Veterinary Immunology and Immunopathology, 168 (3-4), 262-8, doi: 10.1016/j.vetimm.2015.10.014 (2015)18. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54726/54726fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54726/54726fig2.jpg" /> 
Figure 2: Blank wells are important to rule out DNA contamination or interference by the agonist. A commercial source of LPS was added to acellular wells containing RPMI. A large fold change in fluorescence compared with wells containing non-stimulated neutrophils suggests contamination of the agonist with bacterial DNA. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54726/54726fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release at JoVE.com

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

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 ...

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Immunology and Infection

8.1K Views.  Iowa State University. 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.

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

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 ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

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 ...

Neutrophil Functional Assays for Screening Different Signaling Pathways

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

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

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

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

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

Real-Time Microscopy for Studying NETosis Kinetics and Screening Inhibitors

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

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

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

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

Biology

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

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

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

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

Summary

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