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.

Georgia Üniversitesi Kurumsal Değerlendirme Kurulu sağlıklı gönüllülere (UGA # 2012-10769-06) periferik kan toplamak için insan konu çalışmayı onayladı. 5,7,8 Gönüllüler kan beraberlik önce gerekli bilgilendirilmiş onam formunu imzaladı. Bu makalede yapılan araştırmalar Helsinki Bildirgesi&#39;nin insan denekler üzerinde tıbbi araştırmalar için etik kurallarına uygun olduğunu. Periferik İnsan Kanı adlı Nötrofiller 1. izolasyonu (Şekil 1) Not: Periferik kan nötrofillerin izole edilmesi için çeşitli yollar vardır. Aşağıdaki protokol, bir imkanı sağlar. Bu protokol, dış uyarım üzerine NET&#39;leri salabilen aktif olmayan, insan nötrofil sayıda verir. 40 ml delme ile sağlıklı gönüllülerden elde edilen tam kan kullanın. 7,9 <ol><li> iki adet 50 ml konik tüpler içine kısım 20 ml kan. 10 mi% 6 Dekstran ekleyin. hafifçe karıştırın. </li><li> 20 dakika transferi lökosit ric sonratemiz 50 ml&#39;lik konik tüpler içine herhangi peletlenmiş kırmızı kan hücrelerinin alarak ve steril PBS ile doldurmak olmadan h üst faz. </li><li> 400 x g&#39;de 10 dakika santrifüje. 4 ml süspanse pelet PBS steril. </li><li> % 85 5-adım Percoll degrade hazırlayın -80% -75% -70% -65% iki 15 ml konik tüpler ve katman 2 ml lökosit süspansiyonu her degrade üstünde. </li><li> frenler kapalı olarak 30 dakika boyunca Santrifüj 800 xg. </li><li> tüm hücreler% 75 /% 80 tabakalarında (nötrofilleri) ve% 70 /% 75 arayüzü toplayın. </li><li> PBS içinde iki kez (350 xg, 10 dakika, RT) hücre yıkayın ve bir hemasitometre kullanarak hücreleri saymak. </li></ol> Bir Mikroplaka florimetrede Kullanarak Hücre dışı DNA Yayın Kinetiği 2. Ölçüm Not: Bu yöntem, bir zara-geçirmeyen DNA bağlayıcı 96 oyuklu bir mikro-plaka biçiminde DNA serbest gösterge boya (Şekil 2) arasında floresan değişikliklerin ölçülmesini sağlar. <ol><li> + Deney ortamı içinde nötrofil bir süspansiyonu (HBSS hazırlanması2 x 10 6 hücre / ml&#39;lik bir konsantrasyonda,% 1 (h / h) otolog serum + 5 mM glükoz). </li><li> Zarın geçirgenlik göstermediği bir DNA bağlayıcı boya 5 umol / L ekleyin. hafifçe karıştırın. </li><li> Kısım İnsan nötrofilleri (50 ul / oyuk) 96 çukurlu mikrolevhaların siyah şeffaf dibine. Emin deney ortamı kuyunun tüm alt kapsar emin olun. Değilse, yan hafifçe plaka dokunun. </li><li> 37 ° C&#39;de 10 dakika süreyle plaka içeren nötrofillerin ısıtın. </li><li> Bu sırada nihai konsantrasyonlarının iki katı uyaranların çözümler hazırlamak kullanılmak üzere (37 ° C sıcak bir deney ortamı içinde). Pozitif NET kontrolü maksimum NET salınımını tetiklemek için 4 saat boyunca 100 nM forbol-miristat-asetat (PMA) insan nötrofilleri kullanmak spontan NET oluşumu düşük 5,8 kalır. 4 saat boyunca 5,8 PMA uyarımı maksimal NET salınımını sağlar. </li><li> Ölçme (4 saat, 37 ° C, uyarma için 530 nm ve emisyon 590 nm) için mikroplak florimetresi hazırlayın. &lt;/ Li&gt; <li> 50 ul nötrofil süspansiyonlar 50 ul uyaran çözeltisi ilave edilerek nötrofillerin uyarırlar. Maksimal bir DNA salma sinyalinin ölçülmesi de bir 0.5 ug / ml saponin kullanın. </li><li> okuyucusunda plaka koyun ve hiçbir çalkalanarak her 2 dakikada 4 saat floresan değişiklikleri ölçer. </li><li> Veri analizi için, zamanla floresan normalize artışlar hesaplar. <ol><li> Saponin dahil olmak üzere tüm kuyuları için son nokta değerleri çıkarınız, taban floresans (Şekil 2A, B). saponin floresan artış en yüksek sinyal olmalıdır. Bu &quot;olarak maksimum DNA serbest&quot; (Şekil 2A) olarak ifade edilir. </li><li> maksimal DNA salınımı ile bilinmeyen numunelerin floresan artışlar bölerek bilinmeyen örneklerinde DNA sürümü yüzdelerini hesaplayınız. </li><li> Çoğaltır ortalama sonuçları ve &quot;maksimal DNA sürüm%&quot; (Şekil 2C) olarak sunuyoruz. </li></ol></li></ol> NET Formu 3. kantitasyonuMPO-DNA ve HNE-DNA ELISA tahlilleri ile tirme Not: laboratuvarımızda Bu tahliller, modifiye edilmiş (MPO-DNA) ya da kuruldu (HNE-DNA) MPO-DNA ve HNE-DNA kompleksleri seviyeleri ölçülerek NET oluşumu miktarının 5. <ol><li> Ceket 96 oyuklu yüksek bağlama gece boyunca antikorlarla (50 ul / kuyucuk) kapasitesi ELISA plakaları: Anti-MPO (1: 2000 seyreltme) ya da anti-HNE (1: 2,000). </li><li> PBS ile yıkayın ELISA plakaları, üç kez (200 ul / oyuk) ve oda sıcaklığında 2 saat süre ile% 5 BSA ve% 0.1 insan serum albümini ile bloke (200 ul / oyuk). PBS ile üç kez yıkanır. </li><li> Tohum 96 oyuklu mikro / göz 100 ul deney ortamı içinde 100,000 hücre yoğunluğunda nötrofiller ve NET oluşumunu uyarır. hücreleri inkübe: 37 ° C 4 saat. </li><li> iyice karıştırın, nötrofil Süpernatantları 2 U / ml DNaz ekleyerek sınırlı DNase sindirim gerçekleştirin. Oda sıcaklığında tutun. </li><li> 11 ul 25 mM EGTA (nihai konsantrasyon 2.5 mM) eklenerek 15 dakika sonra DNase durdurun. İyice karıştırın. SU toplayınTemiz mikrofuj tüpleri içerisinde pernatants. Spin örnekleri oda sıcaklığında 5 dakika boyunca 300 xg&#39;de hücre enkaz kurtulmak için. Temiz mikrofuge&#39;de tüpler içine onların süpernatantlar aktarın. hazır olana kadar buz üzerinde saklayın. </li><li> önceden hazırlanmış &quot;NET standart&quot; bir kısım kullanın. Not: NET standardı en az 5 farklı, bağımsız insan vericilerden elde edilen PMA ile uyarılmış nötrofil DNaz ile sindirilmiş yüzer bir karışımıdır. <ol><li> , Uzun bir süre aynı standart kullanmak toplamak ve her donörden nötrofil süpernatantlar büyük bir ses bölmeyin için. Örneğin PMA ile uyarılan nötrofil DNaz ile sindirilmiş yüzer 2 ml toplama 200 alikotları 200 ELISA plakaları için yeterli (10 ul her) neden olur. NET standart karakterize eden veriler, Şekil 5&#39;de görülmektedir. </li><li> 4 saat boyunca 100 nM PMA insan nötrofilleri uyarır ve DNaz adımları 3,4-3,5 göre kendi süpernatanlanna sindirmek geçerlidir. </li><li> Toplama, havuz alikotu (10 ul) ve serbestze (-20 ° C), nötrofil süpernatanlar. </li><li> en az beş ayrı donörlerden elde edilen bir kısım / donör çözülme, &quot;standart NET&quot; hazırlamak için, bunları karıştırın ve buz üzerinde saklayın. </li><li> çözülmüş örnekleri atın ve bir sonraki deney için taze olanları kullanın. </li><li> PBS + EGTA NET-standart 2 seri seyreltme: 1 hazırlayın. </li></ol></li><li> DNase1-sindirilmiş örnekleri (standart ve bilinmeyen süpernatantlar) PBS + EGTA içinde 20-kat seyreltilir ve yakalama antikorları ile kaplanmış ELISA plakaları üzerinde bunları kısım. </li><li> 4 ° C&#39;de gece boyunca ELISA inkübe edin ve PBS ile uygun üç kez yıkayın. </li><li> Algılama antikoru solüsyonu halinde (100 ul / oyuk): turp peroksidaz konjuge anti-DNA antikoru (1: 500, fare). Karanlıkta 1 saat süreyle inkübe edin. </li><li> PBS ile dört kez yıkamadan sonra, TMB peroksidazı substrat ekleyin: 100 ul / oyuk, 30 dak. Mavi bir renk peroksidaz aktivitesi (mevcut NET) için bir göstergesidir. </li><li> de 100 ul / 1 M HCI ilave ederek reaksiyonu durdurun.mavi çözümler sarı (Şekil 5A) dönecek. </li><li> bir mikro-fotometre kullanılarak 450 nm&#39;de absorbansı okumak. </li><li> Veri analizi için, NET standardına göre MPO-DNA veya HNE-DNA kompleksleri miktarını hesaplayın. <ol><li> bütün örneklerden, deney ortamı GEÇMİŞİ optik yoğunluk değeri çıkarın. </li><li> Nispi net içeriği (Y-ekseni) (Şekil 5A) karşı seyreltilmiş standart numunelerin absorbans değerlerine (X-ekseni) çizilir. </li><li> Bu standart eğrinin nonsaturated aralığını kullanarak (Şekil 5A) (kullanılan yazılım en iyi üstel uyum kullanın) bir trend çizgisi kurmak. Bu eğilim çizgisinin denklemi NET&#39;ler miktarda nicel OD değerlerinin dönüşüm belirler. </li><li> NET-standart (Şekil 5A) yüzdesi olarak bilinmeyen numunedeki NET&#39;ler miktarda verecektir 3.13.3 den trend çizgisi denklemi içine bilinmeyenler ölçülen OD değerlerini yerleştirin. </li><li> Hesaplamakçoğaltır (üç kez) ortalamaları ve (Şekil 5C) &quot;MPO-DNA veya HNE-DNA kompleksleri (standardın%) miktarı&quot; olarak son verileri sunmak. </li></ol></li></ol>

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	<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. Exp. , (2010).</li><li>Rada, B., 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=Pyocyanin-enhanced+neutrophil+extracellular+trap+formation+requires+the+NADPH+oxidase.">Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase.</a> PLoS One. 8, e54205 (2013).</li><li>Neumann, 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=The+antimicrobial+peptide+LL-37+facilitates+the+formation+of+neutrophil+extracellular+traps.">The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps.</a> Biochem J. 464, 3-11 (2014).</li><li>Leon, S. A., Shapiro, B., Sklaroff, D. M., Yaros, M. 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. 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=Elevated+cell-free+serum+DNA+detected+in+patients+with+myocardial+infarction.">Elevated cell-free serum DNA detected in patients with myocardial infarction.</a> Clin Chim Acta. 327, 95-101 (2003).</li></ol>

The authors have nothing to disclose.

NET&#39;ler NET&#39;ler edebiyat sürekli, biyoloji, mekanizma ve yönetmelik rollerine ilişkin birkaç önemli soru belirsizliğini koruyor onların keşfinden bu yana geçen on yıllar içinde genişleyen olmasına rağmen patojenleri öldürmek. 1 nötrofil hangi büyüleyici yeni bir mekanizma temsil etmektedir. Uygun metodoloji NET&#39;leri, bu çok benzersiz antimikrobiyal mekanizma ölçmek için geliştirilmiş olmalıdır. Bu makalede, bir yüksek verimli bir şekilde netler ölçmek için kullanıl...

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.

Bu yazıda rakamlar. Nötrofil izolasyon, deneysel prosedürler ve veri analizi açıklama ile mevcut temsilcisi sonuçları yöntemi tarif 1 insan nötrofil hazırlık ardışık adımları göstermektedir. Bu protokol nötrofil izolasyon tek olası yolu temsil eder. Bu stimülasyon üzerine NET&#39;leri salabilen nötrofillerin istirahat büyük miktarda verir. Floresan bazlı DNA salınım testi nasıl çalıştığını 2 gösteriler...

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.

Ekstraselüler DNA Yayın Yüksek Verimli Ölçme ve İnsan Nötrofiller Kantitatif NET Oluşumu<em&gt; İn 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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Research

JoVE Journal

Immunology and Infection

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

13.0K Görüntüleme.  University of Georgia. High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

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

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

2-foton Mikroskop kullanarak Enfekte Akciğerler Nötrofiller Fagositoz ve NET-oluşumu Doğrudan Gözlem

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

İmmünoloji ve Enfeksiyon

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

İmmün Yanıt ve Uyku içinde kantitatif ölçümü<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

T Hücreleri İçin Kantitatif Yüksek throughput Tek hücreli Sitotoksisite Testi

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

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

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

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

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

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Nicel<em&gt; In vitro</emStatik Koşullarında Account İlköğretim İnsan mikrovasküler endotel hücreleri için Nötrofil yapışma Tedbir&gt; Testi

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

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Yüksek verimli Gen Etiketleme içinde<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

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

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Yöntemler lipid Nötrofiller değişiklikler ve Nötrofil Ekstrasellüler Tuzaklar sonraki oluşumunu incelemek

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

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Boş hücre biyokimyasal Fluorometrik enzimatik tahlil yüksek yoğunluklu Lipoprotein Lipid peroksidasyon yüksek üretilen iş ölçümü için

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

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.

Vitro Köpek nötrofil hücre dışı tuzak oluşumu: Floresans mikroskobu dinamik ve nicel analiz

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

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

Plazma zarı verimliliği memeli hücrelerinde yeniden mühürlemeden yüksek üretilen iş ölçümü

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...