The authors gratefully acknowledge Drs James Roth, William Nauseef and Kayoko Kimura and Mr Tom Skadow for assistance with development of the canine neutrophil protocols. RDG is supported by a Wellcome Trust Fellowship ref: WT093767MA.

Tüm deneyler Iowa State Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu etik izni ile yapıldı. 1. Kan Toplama <ol><li> Şirketinden antikoagülan bir safen, baş ya da boyun damarından kan 9 ml çizin (örneğin, etilendiamintetraasetik asit (EDTA), 1.8 mg K 2 EDTA / ml kan) vacutainers veya EDTA içeren kan tüplerine anında transfer bir şırınga içine. Yavaşça rulo veya kan ve anti-koagülan yeterince karışmasını sağlamak için, kan tüpler ters. </li></ol> 2. Nötrofil İzolasyon <ol><li> 50 ml&#39;lik konik bir tüp içinde, steril, oda sıcaklığında eşit hacimde kan karışımı% 3 özenli bir şekilde ters tarafından dekstran-500 (% 0.9 steril tuzlu su içinde oluşan). Oda sıcaklığında 18-20 dakika süreyle dik ayakta bırakın. artık düşük kırmızı katmanı tarafından kontaminasyon olmadan toplanabilir kadar yeni bir tüp saman renkli üst katman aktarın. th eritrositlerE orijinal tüp atılabilir. </li><li> 4 ° C&#39;de 10 dakika boyunca 500 xg&#39;de süpernatant santrifüjleyin. kapalı çizin ve supernatant atın. El ile 10 mi oda sıcaklığında steril fosfat tamponlu tuzlu su (PBS) içinde hücre pelletini yeniden askıya. bu vorteksleme Bu protokolün her aşamasında yeniden askıya nötrofiller için kullanılmamalıdır edin. El ile yerine aynı zamanda hücre verimi en üst düzeye çıkarmak için protokol boyunca tavsiye edilir süpernatantlar dökülen daha kapalı çizim. </li><li> 50 ml konik tüp, oda sıcaklığındaki bir polisükroz ve sodyum Diatrizoate hücre ayırma çözeltisi (ör Histopaque 1077) 10 ml ilave edilir. Dikkatle hücre ayırma çözümü üzerinde hücre içeren çözüm katmanı. Hücre sayısını azaltabilir, oda sıcaklığına gelmesi olmadığını kullanılarak hücre ayırma çözeltisi edin. </li><li> Fren kapalı olarak oda sıcaklığında 30 dakika boyunca 300 x g&#39;de santrifüjleyin. </li><li> Nötrofiller degrade en alt tabakada olduğu gibi, kapalı çizmek ve uppe atmakplazma ve trombositler, tek çekirdekli bir hücre beyaz dar bant ve yoğunluk gradyan ortamının açık bir tabakanın bir üst tabaka oluşur R3 tabakalar. </li><li> Kalan, eritrositleri lizise uğratmak için, oda sıcaklığında 10 ml su nötrofil pelet yeniden askıya. </li><li> 30 saniye sonra, steril oda sıcaklığında% 1.8 sodyum klorür, 10 ml ekleyerek tonikliğinin geri ve yumuşak ters çevirerek karıştırın. </li><li> 4 ° C&#39;de 5 dakika boyunca 500 x g&#39;de santrifüjleyin. süpernatant çizin ve atın. </li><li> pelet hala kırmızı ise, parçalama adımı tekrarlayın. Pelet beyaz (veya lizis zaten iki kez gerçekleştirilmiştir halinde), yavaşça steril PBS, 10 ml hücrelerin yeniden askıya edin. </li><li> otomatik bir sayaç veya hemasitometre kullanarak hücreleri sayın. Tipik verim kan ml&#39;si başına en az 10 6 hücre vardır. </li><li> 4 ° C&#39;de 5 dakika boyunca 500 x g&#39;de santrifüjleyin. süpernatant çizin ve atın. </li><li> steril oda sıcaklığı PBS içinde istenen konsantrasyona hücrelerin yeniden askıya. for DNA salınım testi ml başına 5 x 10 6 hücre, 2. bölümde tekrar süspansiyon nitelendirdi. </li><li> Arzu edildiği takdirde, hücrelerin küçük bir hacmi ile doğrudan transferi veya cam slayt, bir sitosantrifüj kullanılmıştır. Hücreler kuru ve leke &quot;, üretici talimatlarına göre hızlı bir sabitleme ve boyama kiti kullanılarak izin verin. mikroskop altında incelendiğinde, nötrofil izolasyonu, başarılı ise çekirdekli hücrelerin en az% 95 oranında en az eritrosit bulaşma ile granülositler (nötrofiller, bazofiller, eozinofiller) olmalıdır. </li></ol> 3. DNA Salım Deneyi <ol><li> nötrofil izolasyon hemen sonra, DNA salım deneyi ayarlayın. </li><li> Hücre topaklanma ışık mikroskobu ile stok solüsyon muayenesinde varsa, hemen tahlil kurmadan önce steril 70 mikron steril filtre ile hücre süspansiyonu geçmektedir. </li><li> Steril 96 oyuklu plakanın her bir test kuyucuğuna, 5 x 10 4 hücre / çukur ekleyin; Agonist ve Roswell Park Notfenol kırmızı ve 100-200 ul son hacim olması için% 0.5 ısıyla inaktive edilmiş cenin dana serumu ile takviye edilmiş olmayan arteryel Enstitüsü 1640 (RPMI) ortamında. nötrofil stok çözeltisi PBS eşit hacimde ile ikame edildiği agonist en az iki Boş kaplar içerir. Her agonist için boş kuyu kurma floresan deneyi ile agonist tarafından agonisti veya parazit DNA kontaminasyonu dışlanması önemli olduğunu unutmayın. </li><li> bir karbon dioksit (% 4) kuluçka makinesi içinde, 30 dakika boyunca 39 ° C&#39;de inkübe edin. </li><li> istenen konsantrasyonlar ve hücre geçirmeyen bir nükleik asit boyası agonistler ekleyin. farklı nükleik asit boyaları arasında değişecektir hücre geçirmeyen boyanın optimal konsantrasyonunu edin. agonistleri ortamın eşit hacimde ile ikame edildiği, uyarılmamış kontrol her deneyde dahil edilmelidir. NOT: Bu kuyu arasında tutarlı koşulları sağlamak için kültür ortamı içindeki hacim agonistleri seyreltilmesi tercih edilir. 200 nihai oyuk hacimleri &amp;# 181; l başarıyla kullanılmaktadır ve bu değişmiş ise, iyi hacimleri tüm koşullar için aynı kalmalıdır. Forbol-12-miristat-13-asetat (PMA) (nihai konsantrasyon ≥0.1 uM) ya da platelet aktive edici faktör (PAF) (nihai konsantrasyon ≥31 uM) ve pozitif kontrol olarak da kullanılabilir. Agonistler iki kez, en azından ölçülmelidir. </li><li> 39 ° C&#39;de inkübe edin. 2 saat sonra veya arzu edilen aralıklarda bir mikroplaka okuyucusu kullanılarak floresan ölçülür. Optimum zaman aralığı kullanılan agonistler ile değişecektir unutmayın. NOT: Dalga boyu istihdam boya bağlıdır. </li><li> ortalama floresan hesaplamadan önce boş floresan çıkarın. </li><li> bireyler arasında ve plakalar arasında karşılaştırma sağlamak için, uyarılmamış kuyuların ortalama fluoresans ile uyarılmış kuyuların ortalama floresan bölünmesiyle floresansta bir katlama değişimi hesaplamak. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

sunulan DNA salınım testi dışı DNA için bir kolaylıkla ölçülebilir testtir. Yöntem, diğer türlerdeki NET oluşumunu değerlendirmek için kullanılan benzer teknikler uyarlanmıştır ancak santrifüj hızları agonist konsantrasyonları ve inkübasyon süreleri köpek nötrofil 8,17,18 ile kullanım için bir yöntem optimize etmek için değiştirildi. Benzer düzenlemeler, diğer türler için bir yöntem adapte yapılabilir. deney gibi akış sitometrisi veya görüntü analizi gibi NETosis ?...

70 milyon evcil köpekler 1 yalnız ABD&#39;de bulunmaktadır. Değerli aile üyeleri olarak, bu hayvanlar genellikle kenar tıbbi bakım kesim alırsınız. Onlar bizim ortamı paylaşmak Eşit, çünkü köpekler insan hastalıkları 1 patogenezinde ve tedavisinde içgörü sağlayabilir. Ancak, veterinerlik uygulamaları ya da tam tersi olarak insan tıbbında keşifler çeviri olup, iyice gibi doğuştan gelen bağışıklık yanıtı bile yüksek ölçüde korunmuş sistemlerde türleri varyasyonları karakterize etmek için önemlidir. Köpek ve insan doğuştan gelen bağışıklık sistemi arasındaki farklar örnekleri köpek nötrofil 2 yüksek ifade CD4 arasında; Köpeklerde 3 sitoplazmik flagellin sensörü IPAF fonksiyonel homolog olmaması ve etobur 4 bir kaspaz 1/4 melez ifadesi. Nötrofil dışı tuzaklar (NET) doğuştan gelen bağışıklık 5 nispeten yeni keşfedilen bileşenidir. NET&#39;ler ağı vardırenflamatuar veya enfeksiyöz uyaranlara 6 geniş bir tepki olarak serbest DNA çekirdek ve granül proteinleri s. NET benzeri yapılar tavuk 7, balık 8, yumuşakçalar 9 ve 10 acoelomates dahil olmak üzere birçok türler arasında gösterilmiştir, ancak tür farklılıklar vardır. Örneğin, kemirgen nötrofiller, insan nötrofillerinden daha NETosis uyaranlara yanıt daha yavaş ve daha az yaygın NET&#39;leri 11 oluşturur. NET&#39;ler doğrudan patojenleri 12,13 öldürme dahil olabilir daha tartışmalı mikropları yakalamak ve çoklu türlerden kanıt büyük bir gövde var. Ancak, NET bileşenleri de, doku hasarına geliştirmek 14,15 Otoantijenler olarak trombozu ve hareket teşvik. NET&#39;ler yararlı ve zararlı etkilerini arasındaki denge tür ve ilgi durumda her iki NET&#39;leri araştırılması önemlidir öne farklı hastalıklar ve farklı türler arasında değişebilir. buradauyaran ve köpek nötrofiller tarafından NET&#39;ler salınımını ölçmek için basit bir protokol açıklar. Bu yöntem, nötrofil 16 izole etmek ve diğer türlerde NETosis uyarılması için kullanılanlara benzer, ancak bu tür agonist konsantrasyonunun ve kuluçkalama süresi gibi koşullar, Kanin nötrofiller için optimize edilmiştir. Benzer bir .NET miktar DNA salma tahlili diğer türlerde de tarif edilmiştir, ancak burada sunulan yöntem köpeklerde 8,17,18 için optimize edilmiştir.

<table><tbody><tr><td>Plastic Whole Blood tube with spray-coated K&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>BD</td><td>367835</td><td></td></tr><tr><td>Histopaque-1077</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>Dextran-500</td><td>Accurate chemical and scientific corp.</td><td>AN228410</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>ThermoFisher scientific</td><td>20012043</td><td></td></tr><tr><td>Fetal bovine calf serum, heat inactivated</td><td>ThermoFisher scientific</td><td>10100139</td><td></td></tr><tr><td>RPMI Media 1640, without phenol red or L-glutamine</td><td>ThermoFisher scientific</td><td>32404-014</td><td>Should be free from phenol red</td></tr><tr><td>96 well flat bottomed sterile polystyrene plate</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich</td><td>P1585</td><td></td></tr><tr><td>Platelet activating factor</td><td>Sigma-Aldrich</td><td>P4904</td><td></td></tr><tr><td>SYTOX Green Nucleic Acid Stain</td><td>ThermoFisher scientific</td><td>S7020</td><td></td></tr><tr><td>Synergy 2 Multi-Mode Reader</td><td>BioTek</td><td>NA</td><td></td></tr></tbody></table>

a simple fluorescence assay for quantification of canine neutrophil extracellular trap release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Bu protokol kullanılarak, pozitif kontroller, PMA ve PAF ile nötrofilleri stimüle sonra floresan kuvvetli bir kat değişiklik olması gerekir. Uyarılmamış hücrelere (dağılım 2,0-5,8, s = 5 köpek) 18 ile karşılaştırıldığında floresanstaki ortalama 4.0 kat bir artış ile 1 saat sonuçlar için 31 uM PAF Şekil 1B, köpek nötrofillerin uyarılması gösterildiği gibi. 0.1 uM (Şekil 1A) PMA 2 saat kadar floresansta bir art?...

Watch this Scientific Journal Video about A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release at JoVE.com

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

Nötrofil dışı tuzaklar (NET) DNA, histon ve nötrofil proteinlerin ağlardır. Doğuştan gelen bağışıklık tepkisinin bir bileşeni olmasına rağmen, ağ otoimmün ve tromboz implike edilmiştir. Bu protokol, köpek nötrofil izolasyon ve bir mikroplaka flüoresans deneyi kullanılarak NET&#39;ler ölçümü için basit bir yöntem tarif eder.

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 ...

a-simple-fluorescence-assay-for-quantification-canine-neutrophil

Research

JoVE Journal

Immunology and Infection

8.0K Görüntüleme.  Iowa State University.  Nötrofil dışı tuzaklar (NET) DNA, histon ve nötrofil proteinlerin ağlardır. Doğuştan gelen bağışıklık tepkisinin bir bileşeni olmasına rağmen, ağ otoimmün ve tromboz implike edilmiştir. Bu protokol, köpek nötrofil izolasyon ve bir mikroplaka flüoresans deneyi kullanılarak NET'ler ölçümü için basit bir yöntem tarif eder. 

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Nötrofil Ekstrasellüler Tuzaklar: Onları nasıl oluşturun ve görselleştirin için

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

İmmünoloji ve Enfeksiyon

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Hücresel ve Biyofilmler Ekstrasellüler Bileşenleri Eşzamanlı Kantitasyonu

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

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.

İnsan Nötrofillerinde Nötrofil Hücre Dışı Tuzakların Oluşumunu Ölçmek için Gerçek Zamanlı Yüksek Verim Tekniği

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

Farklı Sinyal Yollarının Tizlenmesi için Nötrofil Fonksiyonel Tahliller

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

Yazar Spotlight: NeutroFun Ekran Protokolü ile Nötrofil Fonksiyon Değerlendirmesinde Hız, Maliyet ve Doğruluğun Dengelenmesi 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

Nötrofil fonksiyonuna hızlı bir genel bakış için bir dizi tarama tekniği

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

Köpek Nötrofil Ekstrasellüler Trap Release Niceleme için Basit Floresan Testi

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Hücresel ve Biyofilmler Ekstrasellüler Bileşenleri Eşzamanlı Kantitasyonu

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

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

İnsan Nötrofillerinde Nötrofil Hücre Dışı Tuzakların Oluşumunu Ölçmek için Gerçek Zamanlı Yüksek Verim Tekniği

İnsan Nötrofillerinde Nötrofil Hücre Dışı Tuzakların Oluşumunu Ölçmek için Gerçek Zamanlı Yüksek Verim Tekniği

İnsan Nötrofillerinde Nötrofil Hücre Dışı Tuzakların Oluşumunu Ölçmek için Gerçek Zamanlı Yüksek Verim Tekniği

Nötrofil fonksiyonuna hızlı bir genel bakış için bir dizi tarama tekniği