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 forsøkene ble utført med etisk tillatelse fra Iowa State University Institutional Animal Care og bruk komité. 1. Blodprøvetaking <ol><li> Tegn 9 ml blod fra en saphena, cefalisk eller halsvenen direkte inn i antikoagulant (som etylendiamintetraeddiksyre (EDTA), 1,8 mg K 2 EDTA / ml blod) vacutainers, eller inn i en sprøyte med umiddelbar overføring til EDTA-inneholdende blod rør. Forsiktig rulle eller invertere blod rør for å sikre tilstrekkelig blanding av blod og antikoagule...

<ol>
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DNA utgivelsen analysen som presenteres er en lett kvantifiserbare analyse for ekstracellulært DNA. Metoden ble tilpasset fra tilsvarende teknikker som brukes for å vurdere NET dannelse i andre arter, men sentrifugehastigheter, agonist konsentrasjoner og inkubasjonstider har blitt endret for å optimalisere fremgangsmåten for bruk med canine neutrofiler 8,17,18. Lignende justeringer kan foretas for å tilpasse metoden for andre arter. Analysen er enkel og billig sammenlignet med andre metoder for å måle ...

Det er over 70 millioner kjæledyr hunder i USA alene en. Som verdsatte familiemedlemmer, disse dyrene ofte får cutting edge medisinsk behandling. Like fordi de deler vårt miljø, kan hunder gi innsikt i patogenesen og behandlingen av menneskelig sykdom 1. Men om sette funn i humanmedisinen til veterinær behandling eller vice versa, er det viktig å grundig karakterartsvariasjoner selv i sterkt konserverte systemer slik som det medfødte immunrespons. Eksempler på forskjeller mellom hund og menneske medfødte immunsystemet omfatter høyt uttrykk CD4 på hunden nøytrofile 2; fraværet av en funksjonell homolog av det cytoplasmatiske flagellin sensoren IPAF i hunder 3 og ekspresjon av et caspase 1/4 hybrid i rovdyr 4. Nøytrofile ekstracellulære feller (NETS) er en relativt nylig oppdaget komponent av medfødt immunitet 5. NET er nettverkss av DNA, nukleære og granulære proteiner utgitt i respons til et bredt spekter av inflammatoriske eller infeksiøse stimuli 6. Nettliknende strukturer har blitt vist i mange arter inkludert kyllinger 7, 8 fisk, bløtdyr 9 og acoelomates 10, men det er artsvariasjoner. For eksempel murine nøytrofile svare saktere å NETosis stimuli enn humane neutrofiler, og danner mindre diffuse NET 11. Det er en stor mengde bevis fra flere arter som NET entrap mikrober, og mer kontroversielt kan være direkte involvert i å drepe patogener 12,13. Men NET komponenter også forbedre vevsskade, fremme trombose og fungere som autoantigener 14,15. Balansen mellom de gunstige og skadelige effekter av nøter kan variere mellom ulike sykdommer og ulike arter, noe som tyder på at det er viktig å undersøke NET både i art og tilstand av interesse. her er vibeskrive en enkel protokoll for å indusere og måling av utslipp av Nets av hjørnetann nøytrofile. Denne metoden er lik de som brukes for å isolere neutrofiler 16 og indusere NETosis i andre arter, men forhold som agonist-konsentrasjon og inkubasjonstid har blitt optimalisert for canine nøytrofiler. En lignende NET kvantifisering DNA frigjøringsanalyse har også blitt beskrevet i andre arter, men fremgangsmåten som presenteres her er også optimert for hunder 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 hjelp av denne protokollen, bør det være en sterk gangers endring i fluorescens etter å stimulere nøytrofiler med de positive kontroller, PMA og PAF. Som illustrert i figur 1B, stimulering av hjørnetann nøytrofile med 31 mikrometer PAF 1 time resulterer i en gjennomsnittlig 4,0 ganger økning i fluorescens sammenlignet med ikke-stimulerte celler (range 2,0 til 5,8, n = 5 hunder) 18. PMA ved 0,1 um (figur 1A) er en langsommere agonist...

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

Nøytrofile ekstracellulære feller (nett) er nettverk av DNA, histoner og nøytrofile proteiner. Selv om en komponent av den medfødte immunrespons, er garn implisert i autoimmunitet og trombose. Denne protokollen beskriver en enkel metode for canine nøytrofil isolering og kvantifisering av nett ved hjelp av en mikroplate-fluorescens-analyse.

En enkel Fluorescens analyse for Kvantifisering av Canine nøytrofile ekstracellulær felle utløsning

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

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