Name: in vitro canine neutrophil extracellular trap formation: dynamic and quantitative analysis by fluorescence microscopy
Uploaded: 2018-08-24T13:30:00
Description: Watch this Scientific Journal Video about In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy at JoVE.com

Tilsvarende forfatteren blev finansieret af Morris Animal Foundation (D15CA-907). Undersøgelsen blev støttet af midler fra University of California, Davis, Center for Equine sundhed og Center for Companion Animal Health (2016-24-F). Forfatterne vil gerne anerkende Geena Ng for hendes hjælp med tal og Nghi Nguyen for hendes hjælp med videoen.

Alle metoder, der beskrives her blev godkendt af det institutionelle Animal Care og brug udvalg på University of California, Davis (protokol nummer: 18338).
1. indsamling
<ol><li>Trække 10 mL af blodet fra enten cephalic eller jugularis venen ved hjælp af en 21 G nål ved sprøjte aspiration.</li>
	<li>For at undgå overdreven shear stress, fjerne nål fra sprøjten inden flytningen blod ind i røret indeholdende natrium heparin (75 USP). Forsigtigt invertere rør et par gange at sikre ti...

<ol>
	<li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nature Reviews Immunology. 6 (3), 173-182 (2006).</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=Beneficial+suicide:+why+neutrophils+die,+to+make+NETs.">Beneficial suicide: why neutrophils die, to make NETs.</a> Nature Reviews Microbiology. 5 (8), 577-582 (2007).</li><li>Kaplan, M. 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A., Goggs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+plasma+cell-free+DNA+concentrations+in+dogs+with+sepsis,+trauma,+and+neoplasia.">Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia.</a> Journal of Veterinary Emergency and Critical Care. 27 (3), 307-314 (2017).</li><li>Jeffery, U., Gray, R. D., LeVine, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Fluorescence+Assay+for+Quantification+of+Canine+Neutrophil+Extracellular+Trap+Release.">A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release.</a> J Vis Exp. 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Vi præsenterer her en protokol for at observere ændringer i kerner kropsbygning og cfDNA udgivelse i levende canine neutrofiler ved hjælp af både en celle permeant farvestof og en celle impermeant farvestof. Den største fordel ved denne analyse er, at det giver mulighed for real-time detektering af netto dannelsen af høj opløsning mikroskopi i live neutrofiler uden celle fiksering, derfor, at give et simpelt og værdifuldt redskab for observation i vitro netto dannelse. Da denne analyse ikke kræver antis...

Neutrofiler er kortlivede granulocytter ansvarlig for den indledende forsvar mod invaderende patogener. Neutrofiler, rekrutteret til infektionsstedet, fjerne mikroorganismer ved fagocytose, degranulering og generation af reaktive ilt arter (ROS)1. Bakterier eller endotoksiner dekoreret neutrofiler release neutrofile ekstracellulære fælder (net), sammensat af ekstracellulære kromatin med histoner og granulerede proteiner som elastase og myeloperoxidase (MPO)2. Selv om NETs har uundværlig antimikrobielle egenskaber, tyder øge eksperimentel og klinisk dokumentation på, at nidkære netto dannelse under sepsis kan føre til flere organ dysfunktion og død3,4, 5 , 6.
Fordi net kan spille en lignende patofysiologiske rolle i hunde, kan terapeutiske indgreb, der enten forhindre eller mindske netto dannelse tjene som roman behandling strategier i septisk dyr. Derfor er der behov for en pålidelig teknik til at vurdere og bestemme NETosis og netto komponenter i hunde. NETTO komponenter, herunder celle-gratis DNA (cfDNA) og nucleosomes er tidligere blevet evalueret i canine neutrofile og plasma fra kliniske hunde7,8,9. Ved hjælp af fluorescens assays, fundet Goggs og Letendre at septisk hunde have højere niveauer af cfDNA end sunde hunde8. Selv om disse teknikker er meget objektive og kvantitative, er måling af cfDNA og nucleosomes som markører for NETosis uspecifikke, da de kan være afledt af nekrotiske celler end NETosing neutrofiler. Her beskriver vi en teknik, der udnytter Fluorescens mikroskopi for at undersøge adfærd af levende NETosing neutrofiler. Vi detalje også en modificeret protokol bruger dobbelt-immunolabeling til subjektivt kvantificere redskaber og deres komponenter såsom MPO og citH3 i canine neutrofiler10.

<table>
	<tbody>
		<tr>
			<td>Dextran from Leuconostoc spp.</td>
			<td>Sigma</td>
			<td>31392</td>
			<td>Molecular weight 450,000 &#8211; 650,000</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Life Sciences</td>
			<td>17144002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>A1285801</td>
			<td>With divalent cations</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>14190136</td>
			<td>Without divalent cations</td>
		</tr>
		<tr>
			<td>E. coli O55:B5</td>
			<td>InvivoGen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Orange Fluorescent Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S11368</td>
			<td>5 mM in DMSO; stains in cells with permeable membranes</td>
		</tr>
		<tr>
			<td>SYTO Green 16 Fluorescent Nucleic Acid Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S7578</td>
			<td>1 mM in DMSO; stains in cells with intact membranes</td>
		</tr>
		<tr>
			<td>Surface-Amps NP-40</td>
			<td>Pierce</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine coated coverslips</td>
			<td>neuVitro</td>
			<td>H-12-pdl</td>
			<td>12 mm diameter</td>
		</tr>
		<tr>
			<td>Anti-citrullinated histone H3 antibody</td>
			<td>Abcam</td>
			<td>Ab5103</td>
			<td></td>
		</tr>
		<tr>
			<td>Unconjugated goat anti-rabbit Fab fragments</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-007-003</td>
			<td>Specificity: IgG (H+L)</td>
		</tr>
		<tr>
			<td>Anti- Myeloperoxidase antibody</td>
			<td>Dako</td>
			<td>A0398</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-Diamidino-2-phenylin (DAPI)</td>
			<td>Life Technologies Corporation</td>
			<td>D1306</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro canine neutrophil extracellular trap formation: dynamic and quantitative analysis by fluorescence microscopy

Som reaktion på invaderende patogener, frigive neutrofiler neutrofile ekstracellulære fælder (net), som er ekstracellulære netværk af DNA dekoreret med histoner og antimikrobiel proteiner. Overdreven netto dannelse (NETosis) og citH3 udgivelse under sepsis er forbundet med flere funktionsforstyrrelser og dødelighed i mus og mennesker, men dens konsekvenser i hunde er ukendt. Heri, beskriver vi en teknik til at isolere canine neutrofiler fra fuldblod for observation og kvantificering af NETosis. Leukocyt-rich plasma, genereret af dextran sedimentering, er adskilt af kommercielt tilgængelige tæthed gradient adskillelse medier og granulocytter indsamlet celle antal og levedygtighed afprøvning. For at overholde real-time NETosis i live neutrofiler, føjes celle permeant og celle impermeant fluorescerende nukleinsyre pletter til neutrofiler aktiveret enten af LPS (LPS) eller phorbol 12-myristate 13-acetat (PMA). Ændringer i nukleare morfologi og netto dannelse er observeret over tid af Fluorescens mikroskopi. In vitro NETosis er yderligere karakteriseret ved co-colocalization af celle-gratis DNA (cfDNA), myeloperoxidase (MPO) og citrullinated Histon H3 (citH3) bruger en modificeret dobbelt-immunolabelling protokol. For at objektivt kvantificere netto dannelse og citH3 udtryk ved hjælp af Fluorescens mikroskopi, redskaber og citH3-positive celler er kvantificeret i en blindet måde ved hjælp af tilgængelige software. Denne teknik er en specifik assay til at evaluere in vitro- kapaciteten af canine neutrofiler til at gennemgå NETosis.

Bruger denne protokol af levende celle imaging, kan efterforskere observere nukleare morfologi, plasma membran integritet og tilstedeværelsen af cfDNA i levende neutrofiler. En celle impermeant nukleare farvestof pletter kerner syrer rød i celler med beskadiget cellemembraner. En anden celle-permeant farvestof, etiketter intracellulære nukleinsyrer i levende celler med intakte plasma membraner. Alle intakt neutrofiler, uanset deres behandlinger, der skal stå grøn og udviser de karakt...

Watch this Scientific Journal Video about In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy at JoVE.com

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

Vi beskriver metoder til at isolere canine neutrofiler fra fuldblod og visualisere netto dannelse i live neutrofiler ved hjælp af Fluorescens mikroskopi. Også beskrevet er protokoller at kvantificere netto dannelse og citrullinated Histon H3 (citH3) udtryk ved hjælp af immunofluorescens mikroskopi.

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 ...

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JoVE Journal

Immunology and Infection

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

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Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Kvantitative<em&gt; In vitro</em&gt; Assay til måling neutrofiladhæsion til Aktiverede primære humane mikrovaskulære endotelceller under statiske forhold

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High Throughput Måling af ekstracellulær DNA udledning og kvantitativ NET Formation i humane neutrofiler<em&gt; In vitro</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Måling fagosom pH ved Ratiometrisk fluorescensmikroskopi

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Metoder til at studere Lipidforandringer i neutrofiler og den efterfølgende dannelse af neutrofile ekstracellulær fælder

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

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

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 ...

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 ...