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-co...

<ol>
	<li>Schiffman, J. D., Breen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+oncology:+what+dogs+and+other+species+can+teach+us+about+humans+with+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+canine+leucocyte+antigens:+a+significant+advance+in+canine+immunology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplication+of+the+caspase-12+prodomain+and+inactivation+of+NLRC4/IPAF+in+the+dog.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+mammalian+caspases+reveals+an+important+role+of+gene+loss+in+shaping+the+human+caspase+repertoire.">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/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>Brinkmann, V., 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+extracellular+traps:+is+immunity+the+second+function+of+chromatin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+heterophil+extracellular+traps+(HETs):+novel+defense+mechanism+of+chicken+heterophils.">Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils.</a> Vet Immunol Immunopathol. 129 (1-2), 126-131 (2009).</li><li>Pali&#263;, D., Ostoji&#263;, J., Andreasen, C. B., Roth, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+cast+NETs:+neutrophil+extracellular+traps+are+released+from+fish+neutrophils.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+histones+and+DNA+traps+in+invertebrate+immunity:+evidences+in+Crassostrea+gigas.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invertebrate+extracellular+phagocyte+traps+show+that+chromatin+is+an+ancient+defense+weapon.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+double-edged+swords+of+innate+immunity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+by+neutrophil+extracellular+traps:+fact+or+folklore?.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase+associated+with+neutrophil+extracellular+traps+is+active+and+mediates+bacterial+killing+in+the+presence+of+hydrogen+peroxide.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis:+tangled+up+in+NETs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+from+patients+with+rheumatoid+arthritis+target+citrullinated+histone+4+contained+in+neutrophils+extracellular+traps.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+neutrophils+from+venous+blood.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+conventional+protein+kinase+C+(PKC)+is+critical+in+the+generation+of+human+neutrophil+extracellular+traps.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dogs+cast+NETs+too:+Canine+neutrophil+extracellular+traps+in+health+and+immune-mediated+hemolytic+anemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neutrophil+apoptosis+and+lifespan+in+health+and+disease.">Regulation of human neutrophil apoptosis and lifespan in health and disease.</a> J Cell Death. 7, 15-23 (2014).</li></ol>

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 f...

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 border="0" cellpadding="0" cellspacing="0" width="546">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:204px;">Plastic Whole Blood tube with spray-coated K2EDTA</td>
			<td style="width:215px;">BD</td>
			<td style="width:64px;">367835</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Histopaque-1077</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran-500</td>
			<td>Accurate chemical and scientific corp.</td>
			<td>AN228410</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>ThermoFisher scientific</td>
			<td>20012043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine calf serum, heat inactivated</td>
			<td>ThermoFisher scientific</td>
			<td>10100139</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 height="20">
			<td height="20" style="height:20px;">96 well flat bottomed sterile polystyrene plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phorbol 12-myristate 13-acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P1585</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platelet activating factor</td>
			<td>Sigma-Aldrich</td>
			<td>P4904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SYTOX Green Nucleic Acid Stain</td>
			<td>ThermoFisher scientific</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 fl...

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 ...

The overall goal of this experiment is to isolate quiescent neutrophils and measure their DNA release in response to agonists. This method can help answer key questions in immunology and veterinary medicine such as, what is the neutrophil response to various agonists of neutrophil extracelluar trap induction or cell death? The main advantage of this techinique is that it allow the rapid screening of various agonists and inhibitors of neutrophil extracellular trap formation.Begin by inserting a 21-gauge butterfly needle into the cephalic vein of a healthy canine donor then attach a three-milliliter syringe and obtain three three-milliliter blood samples, immediately transferring the samples into uncapped EDTA-containing blood tubes. Take care that the venipuncture is atraumatic and that the blood flow is good, as a poor blood draw will result in a poor neutrophil yield. After filling each tube, invert the tubes to ensure an adequate mixing of the blood and anti-coagulant.Pool the blood in a 50-milliliter conical tube. Mix the samples with an equal volume of sterile room-temperature 3%Dextran 500 by gentle inversion and leave the tube upright for 18 to 20 minutes at room temperature. Then transfer the straw-colored upper layer to a fresh tube until it can no longer be collected without contamination by the lower red layer.Centrifuge the upper layer. Discard the supernatant, manually resuspending the pellet in 10 milliliters of sterile room-temperature PBS. Next, add 10 milliliters of a room-temperature polysucrose in sodium diatrizoate cell separation solution to a 50-milliliter conical tube.Carefully layer the cells over the separation solution. Separate the cells by centrifugation. Discard the upper three plasma and platelet mononuclear cell and density gradient layers.Lyse any of the remaining erythrocytes with 10 milliliters of room-temperature water. After 30 seconds, add 10 milliliters of sterile room-temperature 1.8%sodium chloride to restore the tonicity. Mix the cells by gentle inversion.Now collect the cells by centrifugation. Gently resuspend the white pellet in 10 milliliters of sterile PBS. After counting, centrifuge the cells again and resuspend the pellet at a five times 10 to the sixth cells per milliliter concentration in sterile room-temperature PBS.As appropriate, air-dry a small volume of cells on a glass slide, followed by staining with a rapid fixation and staining kit according to the manufacturer's instructions. If the neutrophil isolation has been successful, at least 95%of the nucleated cells should be granulocytes with minimal erythrocyte contamination as observed under light microscopy. Immediately after the neutrophil isolation, pass the cell suspension through a sterile 70-micron sterile filter and add five times 10 to the fourth cells to the test wells of a 96-well plate containing culture media for a 30 minute incubation at 39 degrees celsius and 4%carbon dioxide.When the cells have attached, add up to a final volume of 100-200 microliters of the agonist of interest in RPMI without phenol red and supplemented with heat-inactivated FCS to the test wells, as well as to two blank wells containing PBS alone. Then add an appropriate cell-impermeable nucleic acid dye to the cells and return the plate to the incubator. After the appropriate incubation period, measure the fluorescence on a microplate reader.A one-hour stimulation of canine neutrophils with platelet-activating factor results in a mean four-fold increase in fluorescence compared with control non-stimulated cells. PMA however, is a slower agonist that does not demonstrate an increased neutrophil fluorescence before two hours of activation, but which ultimately produces a similar fold increase over time. Nucleic acid dye fluorescence is not specific for neutrophil extracellular trap formation, as it will also occur if there is cell death by other mechanisms or if the reagents are contaminated with DNA.For example, in this representative experiment, a commercial LPS preparation produced very high fluorescence in the absence of neutrophils, presumably due to contamination with bacterial DNA. Once mastered, this technique can be completed in approximately six hours if it is performed properly. While attempting this procedure, it's important to remember to handle the neutrophils gently.Do not vortex the cells, and pipette off rather than pour off the supernatants. Following this procedure, other methods, like immunofluorescence microscopy, can be performed to confirm that the DNA released correlates to NETosis. After its development, this technique paved the way for researchers in the field of hematology to explore the interaction of DNA released on coagulation and the effect of novel NET agonists including patient plasma.After watching this video, you should have a good understanding of how to isolate canine neutrophils and measure their response to agonists.

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Research

JoVE Journal

Immunology and Infection

8.0K visualizações.  Iowa State University. The overall goal of this experiment is to isolate quiescent neutrophils and measure their DNA release in response to agonists. This method can help answer key questions in immunology and veterinary medicine such as, what is the neutrophil response to various agonists of neutrophil extracelluar trap induction or cell death? The main advantage of this techinique is that it allow the rapid screening of various agonists and inhibitors of neutrophil extracellular trap formation.Begin by ins...