Name: a simple fluorescence assay for quantification of canine neutrophil extracellular trap release
Uploaded: 2016-11-21T14:00:00
Description: Watch this Scientific Journal Video about A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release at JoVE.com

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 forsøg blev udført med etisk tilladelse fra Iowa State University Institutional Animal Care og brug Udvalg. 1. Blodprøvetagning <ol><li> Tegn 9 ml blod fra en vena, cefal eller halsvenen direkte i antikoagulant (f.eks ethylendiamintetraeddikesyre (EDTA), 1,8 mg K 2 EDTA / ml blod) vakuumbeholdere, eller i en sprøjte med øjeblikkelig overførsel til EDTA-holdige blod rør. Forsigtigt rulle eller invertsukker blodet rør til at sikre tilstrækkelig blanding af blod og antikoagulant....

<ol>
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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. 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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. 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DNA frigivelsesassay fremlagt er en let kvantificerbar assay for ekstracellulært DNA. Metoden blev tilpasset fra lignende teknikker anvendes til at vurdere NET dannelse i andre arter, men centrifugeringshastigheder, agonist koncentrationer og inkuberingstider er blevet ændret for at optimere metoden til anvendelse med hunde neutrofiler 8,17,18. Lignende justeringer kan foretages for at tilpasse fremgangsmåden til andre arter. Assayet er ligetil og billig sammenlignet med andre metoder til måling NETosis s...

Der er over 70 mio hunde i USA alene en. Som værdsatte familiemedlemmer, disse dyr ofte modtager banebrydende lægehjælp. Lige så fordi de deler vores miljø, kan hunde give indsigt i patogenesen og behandling af sygdomme hos mennesker en. Men hvorvidt oversætte opdagelser i humanmedicin i veterinære behandlinger eller omvendt, er det vigtigt at grundigt karakterisere artsvariationer selv i stærkt konserverede systemer såsom det medfødte immunreaktion. Eksempler på forskelle mellem hunde og menneskers medfødte immunsystem omfatter høj ekspression CD4 på hund neutrofiler 2; fraværet af en funktionel homolog af den cytoplasmatiske flagellin sensor IPAF hos hunde 3 og ekspressionen af en caspase 1/4 hybrid i kødædere 4. Neutrofile ekstracellulære fælder (net) er en relativt nylig opdaget komponent af medfødte immunitet 5. NET er netværks af DNA, nukleare og kornede proteiner frigivet som reaktion på en bred vifte af inflammatoriske eller infektiøse stimuli 6. NET-lignende strukturer er blevet påvist på tværs af mange arter, herunder kyllinger 7, fisk 8, bløddyr 9 og acoelomates 10, men der er artsvariationer. For eksempel murine neutrofiler reagere langsommere til NETosis stimuli end humane neutrofiler, og danner mindre diffuse NET 11. Der er en stor mængde dokumentation fra flere arter, som NET indeslutter mikrober, og mere kontroversielt kan være direkte involveret i drab patogener 12,13. Men NET komponenter også øge vævsskader, fremme trombose og fungere som autoantigener 14,15. Balancen mellem de gavnlige og skadelige virkninger af NET kan variere mellem forskellige sygdomme og forskellige arter, hvilket tyder på at det er vigtigt at undersøge NET både i beskaffenhed og tilstand af interesse. Her er vibeskrive en enkel protokol til at fremkalde og måle frigivelse af NET med hunde neutrofiler. Denne metode svarer til dem, der anvendes til at isolere neutrofiler 16 og inducere NETosis i andre arter, men forhold såsom agonist koncentration og inkubationstid er blevet optimeret til hunde neutrofiler. En lignende NET kvantificering DNA frigivelsesassay er også blevet beskrevet i andre arter men metoden præsenteres her er også optimeret til hunde 8,17,18.

<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.

Ved hjælp af denne protokol, bør der være en stærk fold ændring i fluorescens efter stimulering neutrofiler med de positive kontroller, PMA og PAF. Som illustreret i figur 1B, stimulering af hunde neutrofiler med 31 pM PAF i 1 time resulterer i en gennemsnitlig 4,0-fold stigning i fluorescens sammenlignet med ikke-stimulerede celler (interval 2,0-5,8, n = 5 hunde) 18. PMA 0.1 pM (figur 1A) er en langsommere agonist, der ikke resulterer i ...

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

Neutrofile ekstracellulære fælder (net) er netværk af DNA, histoner og neutrofile proteiner. Selv om en del af den medfødte immunreaktion, er NET impliceret i autoimmunitet og trombose. Denne protokol beskriver en enkel fremgangsmåde til hunde neutrofil isolering og kvantificering af NET bruger mikroplade fluorescens-assay.

A Simple Fluorescens Assay for Kvantificering af Canine Neutrofil Ekstracellulær Trap Frigivelse

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.

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

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

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

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

In vitro Coculture Assay at vurdere Patogen induceret Neutrophil Trans-epitel Migration

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

In Vitro Canine neutrofile ekstracellulære fælde dannelse: Dynamisk og kvantitativ analyse af Fluorescens mikroskopi

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

Kvantificering af Efferocytosis af encellede Fluorescens mikroskopi

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

Automatiseret billedbaseret kvantificering af neutrofile ekstracellulære fælder med 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

En høj overførselshastighed analyse til at vurdere og kvantificere neutrofile ekstracellulære fælde dannelse

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

In vitro stimulation og visualisering af ekstracellulær fælde frigivelse i differentierede humane monocytter-afledte makrofager

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

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Humaniseret Mediator Release Assay som en udlæsning for Allergen Potency

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells ...

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

Kvantificering af myeloperoxidase-DNA og neutrofile elastase-DNA-komplekser fra neutrofile ekstracellulære fælder ved hjælp af en modificeret sandwich-ELISA

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