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>

<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. 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. 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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><tbody><tr><td>Anti-Human Neutrophil Elastase&amp;nbsp; Rabbit Ab&amp;nbsp;</td><td>Calbiochem</td><td>481001</td><td>1:2,000x coated</td></tr><tr><td>Anti-Myeloperoxidase Ab (Rabbit)</td><td>Millipore</td><td>07-496</td><td>1:2,000x coated</td></tr><tr><td>DNase-1</td><td>Roche</td><td>10-104-159-001</td><td>1 &amp;#181;g/ml used for digestion</td></tr><tr><td>20 mM 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&amp;nbsp;</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>100 nM final used</td></tr><tr><td>ELISA Plate</td><td>Greiner bio-one</td><td>655061</td><td></td></tr><tr><td>Conical tubes 15 ml</td><td>Thermoscientific</td><td>339650</td><td></td></tr><tr><td>Conical tubes 50 ml</td><td>Thermoscientific</td><td>339652</td><td></td></tr><tr><td>Percoll (pH 8.5-9.5)&amp;nbsp;</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 ...

high-throughput-measurement-extracellular-dna-release-quantitative

Universidad de Cartagena UDEC

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

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Basitleştirilmiş İnsan Nötrofil Ekstrasellüler Tuzaklar (NET) İzolasyon ve Taşıma

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

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.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Köpek Nötrofil Ekstrasellüler Trap Release Niceleme için Basit Floresan Testi

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

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 ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

NETQUANT Kullanarak Nötrofil Ekstrasellüler Tuzakların Otomatik Görüntü Tabanlı Niceliği

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

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Nötrofil hücre dışı tuzak oluşumu ölçmek ve değerlendirmek için bir yüksek-den geçerek tahlil

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

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

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

Nötrofil Hücre Dışı Tuzaklardan Miyeloperoksidaz-DNA ve Nötrofil Elastaz-DNA Komplekslerinin Modifiye Sandviç ELISA Kullanılarak Miktarının Belirlenmesi

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

Çift Boyalı Canlı Hücre Analizi Kullanarak NET Oluşumunun Ölçülmesi

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Yazar Spotlight: Çift Renkli Canlı Hücre Görüntüleme Kullanarak Hastalık ve İlaç Taramasında Nötrofil Hücre Dışı Tuzakların Ölçülmesi

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Ekstraselüler DNA Yayın Yüksek Verimli Ölçme ve İnsan Nötrofiller Kantitatif NET Oluşumu

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