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.

Alle Versuche wurden mit ethischen Erlaubnis von der Iowa State University Institutional Animal Care und Use Committee durchgeführt. 1. Blutentnahme <ol><li> Zeichnen 9 ml Blut aus einer Vena, cephalica oder Halsvene direkt in Antikoagulans (zB Ethylendiamintetraessigsäure (EDTA), 1,8 mg K 2 EDTA / ml Blut) Vacutainer oder in eine Spritze mit sofortiger Transfer zu EDTA-haltige Blutröhrchen. Sanft rollen oder die Blutröhrchen invertieren eine ausreichende Durchmischung des Blutes und der Antikoagulans zu gewährleisten. </li></ol> 2. Neutrophilen-Isolation <ol><li> In einem 50 ml konischen Röhrchen, mischen Blut mit einem gleichen Volumen von sterilen Raumtemperatur 3% Dextran-500 (hergestellt in 0,9% steriler Kochsalzlösung) durch vorsichtiges Umdrehen. Lassen Sie aufrecht für 18-20 Minuten bei Raumtemperatur stehen. Übertragen die strohfarbene obere Schicht in ein frisches Röhrchen, bis es nicht mehr ohne Verunreinigung durch die untere rote Schicht gesammelt werden kann. Die Erythrozyten in the kann original Rohr verworfen werden. </li><li> Zentrifuge den Überstand bei 500 xg für 10 min bei 4 ° C. Absaugen und den Überstand verwerfen. das Zellpellet in 10 ml Raumtemperatur steriler phosphatgepufferter Salzlösung (PBS) manuell erneut zu suspendieren. Beachten Sie, dass Verwirbelung sollte zu keinem Zeitpunkt dieses Protokolls zu resuspendieren Neutrophilen verwendet werden. Manuelles Abziehen, anstatt Stände Abgießen auch im gesamten Protokoll wird empfohlen Zellausbeute zu maximieren. </li><li> 10 ml einer bei Raumtemperatur Polysaccharose und Natriumdiatrizoat Zelltrennungslösung (zB Histopaque 1077) zu einem 50 ml konischen Röhrchen. Schicht sorgfältig die Zellen enthaltende Lösung über die Zelltrennlösung. Man beachte, dass unter Verwendung von Zelltrennungslösung, die auf Raumtemperatur äquilibriert nicht hat, kann Zellausbeute reduzieren. </li><li> Zentrifuge bei 300 × g für 30 min bei Raumtemperatur mit der Bremse ab. </li><li> Als Neutrophilen in der untersten Schicht des Gradienten sind, abtrennen und verwerfen die upper 3 Schichten, bestehend aus einer oberen Schicht aus Plasma und Blutplättchen, einen schmalen weißen Band von mononukleären Zellen und eine klare Schicht der Dichtegradientenmedium. </li><li> alle verbleibenden Erythrozyten, resuspendieren die Neutrophilen-Pellet in 10 ml Wasser bei Raumtemperatur zu lysieren. </li><li> Nach 30 Sekunden wieder her Tonus von 10 ml sterilem Raumtemperatur 1,8% Natriumchlorid-Zugabe und durch vorsichtiges Umdrehen mischen. </li><li> Zentrifuge bei 500 × g für 5 min bei 4 ° C. Zeichnen Sie den Überstand ab und verwerfen. </li><li> Wenn das Pellet noch rot ist, wiederholen Sie den Lyseschritts. Wenn das Pellet weiß (oder, wenn die Lyse bereits zweimal durchgeführt worden ist), vorsichtig resuspendieren Zellen in 10 ml sterilem PBS. </li><li> Zählen von Zellen unter Verwendung eines automatisierten Zähler oder einer Zählkammer. Typische Ausbeuten sind mindestens 10 6 Zellen pro ml Blut. </li><li> Zentrifuge bei 500 × g für 5 min bei 4 ° C. Zeichnen Sie den Überstand ab und verwerfen. </li><li> Resuspendieren Zellen auf die gewünschte Konzentration in steriler PBS Raumtemperatur. for die DNA - Freisetzungstest in Abschnitt 2, resuspendieren bei 5 x 10 6 Zellen pro ml beschrieben. </li><li> Falls gewünscht, Transfer direkt ein kleines Volumen von Zellen oder eine Zytozentrifuge auf einem Glasobjektträger verwendet wird. Zellen erlauben zu trocknen und zu Flecken eine rasche Fixierung und Färbung Kit gemäß den Herstellern &#39;Anweisungen. Wenn der Neutrophil Isolation erfolgreich, wenn sie unter dem Mikroskop bei mindestens 95% der kernhaltigen Zellen untersucht, sollte Granulozyten (Neutrophile, Basophile, Eosinophile) mit minimaler Erythrozytenkontamination sein. </li></ol> 3. DNA-Freisetzungstest <ol><li> Stellen Sie die DNA-Freisetzungstest unmittelbar nach Neutrophilen Isolierung auf. </li><li> Wenn die Zelle Verklumpung auf Prüfung der Stammlösung durch Lichtmikroskopie vorhanden ist, übergeben Sie die Zellsuspension durch ein steriles 70 &amp; mgr; m Sterilfilter unmittelbar vor dem Test einrichten. </li><li> Zu jedem Test gut einer sterilen 96 - Well - Platte, fügen Sie 5 x 10 4 Zellen / Vertiefung; Agonisten und Roswell Park Memorial Institute 1640 (RPMI) Medium ohne Phenolrot und mit 0,5% hitzeinaktiviertem fötalem Kälberserum auf ein Endvolumen von 100-200 &amp; mgr; l ergänzt. Fügen Sie mindestens zwei leere Wells pro Agonist, in dem die Neutrophilen-Stammlösung mit einem gleichen Volumen von PBS ersetzt. Beachten Sie, dass die Einrichtung leere Vertiefungen für jede Agonist in Ausschluss DNA-Kontamination des Agonisten oder Störungen durch den Agonisten mit dem Fluoreszenztest wichtig ist. </li><li> für 30 min in einem Kohlendioxid (4%) Inkubator bei 39 ° C inkubieren. </li><li> In Agonisten bei gewünschten Konzentrationen und einer Zelle undurchlässige Farbstoff Nukleinsäure. Notieren Sie sich die optimale Konzentration von Zelle undurchlässige Farbstoff zwischen verschiedenen Nukleinsäure Farbstoffe variieren. Nicht-stimulierte Kontrollen, in denen Agonisten von einem gleichen Volumen an Medium ersetzt werden sollten in jedem Experiment eingeschlossen werden. HINWEIS: Es ist wünschenswert, Agonisten in ähnlichen Volumina Kulturmedium zu verdünnen, konsistente Bedingungen zwischen Vertiefungen zu halten. Schluss gut Volumen von 200 &amp;# 181; l wurden erfolgreich und wenn dies geändert wird, auch Volumina sollten identisch bleiben für alle Bedingungen. Phorbol-12-Myristat-13-Acetat (PMA) (Endkonzentration ≥0.1 uM) oder Platelet Activating Factor (PAF) (Endkonzentration ≥31 uM) kann als positive Kontrollen verwendet werden. -Agonisten Sollten mindestens doppelt gemessen werden. </li><li> Inkubieren bei 39 ° C. Messen Sie Fluoreszenz eines Mikrotiterplattenmessgeräts nach 2 Stunden oder in gewünschten Intervallen verwenden. Beachten Sie, dass optimale Zeitintervalle verwendet Agonisten variieren. HINWEIS: Wellenlänge wird auf Farbstoff abhängig beschäftigt. </li><li> Subtrahiert leere Fluoreszenz vor mittlere Fluoreszenz zu berechnen. </li><li> Damit Vergleich zwischen Individuen und zwischen den Platten, berechnen eine Falte Veränderung der Fluoreszenz durch die mittlere Fluoreszenz der stimulierten Vertiefungen durch die mittlere Fluoreszenz von nicht-stimulierten Vertiefungen geteilt wird. </li></ol>

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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. 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Die Autoren haben nichts zu offenbaren.

Die DNA-Freisetzungstest vorgestellt, ist ein leicht quantifizierbare Assay für die extrazelluläre DNA. Die Methode wurde von ähnlichen Techniken angepasst verwendeten NET Bildung in anderen Spezies zu bewerten, aber Zentrifugation Geschwindigkeiten Agonist - Konzentrationen und Inkubationszeiten verändert wurden 8,17,18 das Verfahren zur Verwendung mit canine Neutrophilen zu optimieren. Ähnliche Anpassungen könnten die Verfahren für andere Arten angepasst werden. Der Test ist einfach und kostengünsti...

Es gibt mehr als 70 Millionen Hunde allein in den USA 1. Wie bewertet Familienmitglieder, oft sind diese Tiere erhalten Schneiden medizinische Versorgung Rand. Ebenso , weil sie unsere Umwelt teilen, können Hunde geben Einblicke in die Pathogenese und Behandlung menschlicher Krankheiten 1. Doch ob Entdeckungen in der Humanmedizin in tierärztliche Behandlungen oder umgekehrt zu übersetzen, ist es wichtig, gründlich Spezies Variationen charakterisieren auch in hochkonservierten Systeme wie die angeborene Immunantwort. Beispiele für Unterschiede zwischen dem Eckzahn und menschlichen angeborenen Immunsystems sind die hohe Expression CD4 auf Hund Neutrophilen 2; das Fehlen eines funktionellen Homolog des zytoplasmatischen Flagellin Sensor IPAF in dogs 3 und die Expression eines Caspase 1/4 Hybrid in Fleischfressern 4. Neutrophile extrazelluläre Fallen (NETs) sind eine relativ kürzlich entdeckte Komponente der angeborenen Immunität 5. NETs sind Netzwerks von DNA, nukleare und granular Proteine in Reaktion auf eine Vielzahl von entzündlichen oder infektiösen Reizen 6 freigegeben. Netzartige Strukturen wurden in vielen Spezies , einschließlich Hühner 7 gezeigt Fisch 8, 9 und Mollusken acoelomates 10, aber es gibt Speziesvariationen. Zum Beispiel reagieren Maus - Neutrophilen langsamer NETosis Stimuli als menschliche Neutrophilen und bilden weniger diffuse NETs 11. Es gibt einen großen Körper von Beweismitteln aus mehreren Arten , die NETs entrap Mikroben und umstrittener direkt bei der Abtötung von Krankheitserregern 12,13 beteiligt sein können. NET - Komponenten auch Gewebeschäden jedoch verbessern, Thrombose und Tat fördern als 14,15 Autoantigene. Die Balance zwischen den nützlichen und schädlichen Wirkungen von NETs können zwischen verschiedenen Krankheiten und verschiedenen Arten variieren, was darauf hindeutet, ist es wichtig, NETs zu untersuchen, sowohl in der Art und Zustand von Interesse. Hier sind wirbeschreiben ein einfaches Protokoll für die Induktion und die Freisetzung von NETs durch Hunde- Neutrophilen zu messen. Dieses Verfahren ist ähnlich zu denjenigen verwendet , um Neutrophile 16 und induzieren NETosis in anderen Arten, aber die Bedingungen , wie beispielsweise Agonist - Konzentration und Inkubationszeit isolieren haben canine Neutrophilen optimiert. Eine ähnliche NET Quantifizierung DNA - Freisetzungstest wurde auch bei anderen Spezies beschrieben worden , aber das hier vorgestellte Verfahren ist auch für Hunde 8,17,18 optimiert.

<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.

Unter Verwendung dieses Protokolls, sollte es eine starke fache Veränderung der Fluoreszenz sein, nachdem Neutrophilen mit den positiven Kontrollen, PMA und PAF stimulieren. Wie dargestellt in 1B, die Stimulation der canine Neutrophilen mit 31 uM PAF für 1 Stunde ergibt sich eine mittlere 4,0-fache Erhöhung der Fluoreszenz im Vergleich zu nicht-stimulierten Zellen (Bereich 2,0 bis 5,8, n = 5 Hunde) 18. PMA bei 0,1 uM (Figur 1A) ist ein lang...

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

Neutrophile extrazelluläre Fallen (NETs) sind Netzwerke von DNA, Histone und Neutrophilen-Proteine. Obwohl eine Komponente der angeborenen Immunantwort sind NETs in Autoimmun- und Thrombose beteiligt. Dieses Protokoll beschreibt ein einfaches Verfahren zur Hunde- Neutrophilen Isolierung und Quantifizierung von NETs Mikrotiterplatten-Fluoreszenztest verwendet wird.

Eine einfache Fluoreszenztest zur Quantifizierung von Canine Neutrophil Extracellular Einklemmschutz

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

a-simple-fluorescence-assay-for-quantification-canine-neutrophil

Research

JoVE Journal

Immunology and Infection

8.0K Aufrufe.  Iowa State University.  Neutrophile extrazelluläre Fallen (NETs) sind Netzwerke von DNA, Histone und Neutrophilen-Proteine. Obwohl eine Komponente der angeborenen Immunantwort sind NETs in Autoimmun- und Thrombose beteiligt. Dieses Protokoll beschreibt ein einfaches Verfahren zur Hunde- Neutrophilen Isolierung und Quantifizierung von NETs Mikrotiterplatten-Fluoreszenztest verwendet wird. 

Serie: 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 Extracellular Traps: So erzeugen und zu visualisieren

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Forschung

Immunologie und Infektion

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

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.

In vitro Coculture Assay zur Bewertung pathogeninduzierter Neutrophiler Transepithelmigration

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

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Humanen neutrophilen Flusskammer Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

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.

Vereinfachte Menschen Neutrophil Extracellular Traps (NETs) Isolierung und Handhabung

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

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-vitro- Eckzahn Neutrophilenzahl extrazelluläre Trap Bildung: Dynamische und Quantitative Analyse von Fluoreszenz-Mikroskopie

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

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantifizierung der Efferocytosis durch einzellige Fluoreszenz-Mikroskopie

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

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.

Automatisierte bildbasierte Quantifizierung von neutrophilen extrazellulären Fallen mit NETQUANT

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.

Ein High-Throughput-Assay zu beurteilen und zu quantifizieren Neutrophilenzahl extrazelluläre Trap Bildung

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

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

In Vitro Stimulation und Visualisierung der extrazellulären Trap-Freisetzung in differenzierten menschlichen Monozyten-abgeleiteten Makrophagen

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...