Name: high throughput measurement of extracellular dna release and quantitative net formation in human neutrophils in vitro
Uploaded: 2016-06-18T13:00:00
Description: Watch this Scientific Journal Video about High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro at JoVE.com

Special thanks to the personnel of the University of Georgia Health Center laboratory for their continuous support of our work on isolating human neutrophils. This work was supported by the start-up fund of Dr. Rada provided by UGA Office of Vice President for Research.

Institutional Review Board vid University of Georgia godkände mänskliga subjektet studie för att samla perifert blod från friska frivilliga (UGA # 2012-10769-06). 5,7,8 Frivilliga undertecknade krävs informerat medgivande innan blodprovstagning. Forskningen utförs i denna artikel är i enlighet med de etiska riktlinjerna för medicinsk forskning på människor av Helsingforsdeklarationen. 1. Isolering av neutrofiler från perifert Människoblod (Figur 1) Obs: Det finns flera sätt att isol...

<ol>
	<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, 1532-1535 (2004).</li><li>Fuchs, T. A., 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=Novel+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> J Cell Biol. 176, 231-241 (2007).</li><li>Kessenbrock, K., 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=Netting+neutrophils+in+autoimmune+small-vessel+vasculitis.">Netting neutrophils in autoimmune small-vessel vasculitis.</a> Nat Med. 15, 623-625 (2009).</li><li>Leffler, J., 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=Degradation+of+neutrophil+extracellular+traps+co-varies+with+disease+activity+in+patients+with+systemic+lupus+erythematosus.">Degradation of neutrophil extracellular traps co-varies with disease activity in patients with systemic lupus erythematosus.</a> Arthritis Res Ther. 15, R34 (2013).</li><li>Yoo, D. G., Floyd, M., Winn, M., Moskowitz, S. M., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NET+formation+induced+by+Pseudomonas+aeruginosa+cystic+fibrosis+isolates+measured+as+release+of+myeloperoxidase-DNA+and+neutrophil+elastase-DNA+complexes.">NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.</a> Immunol Lett. 160, 186-194 (2014).</li><li>Wang, Y., 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=Histone+hypercitrullination+mediates+chromatin+decondensation+and+neutrophil+extracellular+trap+formation.">Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.</a> J Cell Biol. 184, 205-213 (2009).</li><li>Pang, L., Hayes, C. P., Buac, K., Yoo, D. G., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudogout-associated+inflammatory+calcium+pyrophosphate+dihydrate+microcrystals+induce+formation+of+neutrophil+extracellular+traps.">Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps.</a> J Immunol. 190, 6488-6500 (2013).</li><li>Yoo, D. G., 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=Release+of+cystic+fibrosis+airway+inflammatory+markers+from+Pseudomonas+aeruginosa-stimulated+human+neutrophils+involves+NADPH+oxidase-dependent+extracellular+DNA+trap+formation.">Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation.</a> J. Immunol. 192, 4728-4738 (2014).</li><li>Brinkmann, V., Laube, B., Abu Abed, ., Goosmann, U., Zychlinsky, C., 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,+how+to+generate+and+visualize+them.">Neutrophil extracellular traps, how to generate and visualize them.</a> J. Vis. 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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=Free+DNA+in+the+serum+of+cancer+patients+and+the+effect+of+therapy.">Free DNA in the serum of cancer patients and the effect of therapy.</a> Cancer Res. 37, 646-650 (1977).</li><li>Raptis, L., Menard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+and+characterization+of+plasma+DNA+in+normals+and+patients+with+systemic+lupus+erythematosus.">Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus.</a> J Clin Invest. 66, 1391-1399 (1980).</li><li>Chang, C. 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NET representerar en fascinerande ny mekanism genom vilken neutrofiler döda patogener. 1 Även om litteraturen NET har kontinuerligt byggts ut under de senaste tio åren sedan deras upptäckt flera viktiga frågor som rör deras roll i biologi, mekanism och reglering förblir oklart. Lämplig metod måste utvecklas för att mäta NET, detta mycket unika antimikrobiella mekanism. I denna artikel beskrivs metoder som kan användas för att kvantifiera NET i en hög genomströmning sätt. Den första analysen f...

NET formation is a novel mechanism by which neutrophils fight pathogens.1 The core of NETs is nuclear DNA.1 This DNA network is associated with neutrophil granule proteins and histones.1 The main form of NET formation requires the death of neutrophils characterized by chromatin decondensation, disappearance of granular and nuclear membranes, translocation of neutrophils elastase to the nucleus, citrullination of histones and finally the spill of DNA-based NETs.2 NETs entrap and kill a wide variety of microbes and are an essential part of the innate immune weapon repertoire. Uncontrolled NET formation has, however, been linked to numerous autoinflammatory diseases.3,4 Despite their increasingly established relevance, little is known about the mechanism and regulation of NET release. 
Neutrophils dying by releasing NETs are different from apoptotic or necrotic neutrophils.3,5 NET-releasing neutrophils show several features that are characteristic for NET formation. Granule components are associated with DNA in NETs.1 Myeloperoxidase (MPO) and human neutrophil elastase (HNE) are both found in primary granules in resting cells but are translocated to the nucleus to bind to DNA in NETs.1 MPO-DNA and HNE-DNA complexes are specific for NETs, do not occur in apoptotic or necrotic neutrophils.1,3,5 Chromatin decondensation is another feature typical for NETosis.2 NET release also requires citrullination of histones by peptidyl aminidase 4 (PAD4).6 Citrullinated histones are hallmarks of neutrophils that underwent NET release.6
Here three methods are introduced that in combination provide excellent tools to quantitate NETs on a high-throughput scale. The first assay has been used on the field with different changes and quantitates extracellular DNA release in a microplate format. The second and third assays provide confirmation of NETs by measuring NET-specific MPO-DNA and HNE-DNA complexes.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Anti-Human Neutrophil Elastase&#160; Rabbit Ab&#160;</td>
			<td>Calbiochem</td>
			<td>481001</td>
			<td>1:2000X coated</td>
		</tr>
		<tr>
			<td>Anti-Myeloperoxidase Ab (Rabbit)</td>
			<td>Millipore</td>
			<td>07-496</td>
			<td>1:2000X coated</td>
		</tr>
		<tr>
			<td>DNase-1</td>
			<td>Roche</td>
			<td>10-104-159-001</td>
			<td>1ug/ml used for digestion</td>
		</tr>
		<tr>
			<td>20mM EGTA/ PBS</td>
			<td>Sigma-Aldrich</td>
			<td>E3889-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mM EGTA/PBS</td>
			<td>Sigma-Aldrich</td>
			<td>E3889-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell death detection ELISA Anti-DNA POD</td>
			<td>Roche</td>
			<td>11544675001</td>
			<td>1:500X&#160;</td>
		</tr>
		<tr>
			<td>Eon Microplate Spectrophotometer</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 All-in-One microplate software</td>
			<td>Biotek</td>
			<td></td>
			<td>analytical tool (ELISA)</td>
		</tr>
		<tr>
			<td>Sytox orange</td>
			<td>Life Technology</td>
			<td>S11368</td>
			<td>0.2% final concentration/volume</td>
		</tr>
		<tr>
			<td>1 M Hepes</td>
			<td>Cellgro</td>
			<td>25-060-Cl</td>
			<td>Use 10 mM final concentration.</td>
		</tr>
		<tr>
			<td>1 M glucose</td>
			<td>Sigma</td>
			<td></td>
			<td>Use 5 mM final concentration.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Corning</td>
			<td>21-023-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan Flash Ver.2.4.3</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P 8139</td>
			<td>100nM final used</td>
		</tr>
		<tr>
			<td>ELISA Plate</td>
			<td>Greiner bio-one</td>
			<td>655061</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes 15ml</td>
			<td>Thermoscientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes 50ml</td>
			<td>Thermoscientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (pH 8.5-9.5)&#160;</td>
			<td>Sigma</td>
			<td>P 1644</td>
			<td>Sodium Chloride, Sigma, S7653-250G</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Spectrum</td>
			<td>D1004</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Corning Cellgro</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well assay plate black plate clear bottom</td>
			<td>Costar</td>
			<td>3603</td>
			<td></td>
		</tr>
	</tbody>
</table>

high throughput measurement of extracellular dna release and quantitative net formation in human neutrophils in vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

Siffrorna i detta manuskript att beskriva förfarandet för neutrofil isolering, experimentella procedurer, och presentera representativa resultat med förklaring av dataanalys. Figur 1 visar de sekventiella stegen av humant neutrofilt beredning. Detta protokoll representerar endast ett möjligt sätt att neutrofil isolering. Den ger stora mängder av vilande neutrofiler förmåga att frisätta NET vid stimulering. Figur 2 visar hur fluorescensbaserad DN...

Watch this Scientific Journal Video about High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro at JoVE.com

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

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Hög genomströmning Mätning av extracellulärt DNA utsläpp och Kvantitativ NET bildning i humana 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 ...

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

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A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

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Metoder för att studera Lipidförändringarna i neutrofiler och efterföljande bildning av neutrofila Extracellulär Fällor

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

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Cell-free biokemiska Fluorometric enzymatisk analys för High-throughput mätning av lipidperoxidation i hög Density Lipoprotein

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

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