1. Insan kanı PMN İzolasyon Antikoagülan olarak EDTA veya heparin (10 U / ml) ile 24 ml insan kanı hakkında kullanın. <ol><li> 6 ml, 15 ml Falcon tüp Histopaque 1119 ekleyin ve dikkatle üst katmanı 5 ila 7 ml tam kan. </li><li> Frenleme olmadan 20 dakika boyunca 800 xg&#39;de santrifüjleyin. </li><li> Sarımsı ve açık üst katman aspire edin ve atın ve taze Falcon tüpler içine granülositler içeren alt kırmızımsı faz transfer. </li><li> 300 x g. 10 dakika PBS ve santrifüj Falcon tüpleri dolum hücreleri yıkayın </li><li> Bu arada% 100 Percoll, 2 ml 10x PBS, 18 ml Percoll karıştırılarak bir solüsyon hazırlanır. 1x PBS,% 85,% 80,% 75,% 70 ve% Percoll 65 4 ml çözümler hazırlıyoruz. </li><li> Katmanlama 2 ml azalan birbirlerinin üstüne her yüzde bir Percoll degrade ile 2 Falcon tüpleri hazırlayın. </li><li> Santrifüj süpernatantı kaldırdıktan sonra, 4 ml PBS pelet ve tekrar süspansiyon tortulaşmış hücreleri birleştirir. </li><li> Her degradelerin üzerine tabanda dikkatli katman 2 ml. </li><li> Frenleme olmadan 20 dakika boyunca 800 xg&#39;de santrifüjleyin. </li><li> Santrifüj üst katmanı kaldırmak ve% 65 katmanlı en PBMC&#39;ler ile,% 85 katmanlı kadar yeni Falcon tüpler içine beyaz kalan interphases toplamak sonra. </li><li> 300 x g. 10 dakika PBS ve santrifüj ile Falcon tüpleri dolum hücreleri yıkayın </li><li> PBS 2ml supernatant ve tekrar süspansiyon tortulaşmış hücreleri (genellikle&gt;% 95 PMN.) Çıkarın. </li><li> Hemasitometre kullanarak hücreleri saymak. </li></ol> 2. Aktive PMN <ol><li> Steril bir 13 mm yuvarlak cam kapak kayma (1,5) her bir kuyunun içine koyarak 24-iyi bir hücre kültürü tabak hazırlayın </li><li> Tohum 2 x 10 500μl RPMI 5 hücreleri de başına (% 2 insan serum albümin içeren) ve 37 CO 2 inkübatör 1s inkübe ° C. </li><li> Bu arada RPMI 600 nM PMA çözüm hazırlamak ve 100μl de ortalama hücreleri ekleyin. CO 2 inkübatör 4 saat 15 dakika inkübe 37 ° ° C. </li><li> % 4 (son konsantrasyon) PBS içinde çözünmüş paraformaldehid hücreleri saptamak. </li></ol> PMA bir pozitif kontrol olarak hizmet veren ve bugüne kadar NET oluşumunu uyarmak için en güçlü bir ajandır. Alternatif olarak, patojenlerin diğer uyaranlara veya co-ekimi NET indüksiyonu için kullanılabilir. Zaman kursu devam ederken ek paralel olarak hazırladığı numuneler NET oluşumu ilgili durumu kontrol edilebilir. Sabit olmayan hücrelere Sytox Yeşil (Invitrogen) gibi olmayan bir permeant DNA boya eklenirse, sadece ekstraselüler DNA tespit edilecektir. Yeni NET&#39;ler oluşumu şekilde her zaman noktası için, Sytox varlığı bozulmuş olduğundan bir paralel örnek kullanılmalıdır. 3. NET immunolabeling tarafından tespit NET&#39;ler fiksasyon sonra bile çok kırılgan olduğu ve büyük bir dikkatle manipüle edilebilir aksi takdirde çoğunluğu hazırlanması sırasında kaybolan alacak. <ol><li> 24 kuyu kültür plaka takılı hücreleri ile cam kapak fişleri kavisli bir iğne ile kenarında yukarı kaldırarak dikkatlice çıkarın ve ince bir forseps ile ele geçirmek. PBS bir damla ters kapak kayma koyun. Bu bir test tüpü standı kapsayan bir Parafilm kağıda yapılabilir. 3 kez 5 dakika boyunca bu gibi yıkayın. </li><li> Triton X-100 RT az 1 dakika hücreleri permeabilize için% 0.5 &#39;lik bir düşüş aynı şekilde kapak fişleri inkübe edin. 1 dakika için 3 kez PBS içinde yıkayın. </li><li> Parafilm ve ıslak mendil ile nemli bir oda hazırlayın. Lay kapağı engelleme tampon (% 5 eşek serum) bir damla baş aşağı kayar ve 37 ° 30 dakika inkübe ° C. </li><li> Tampon engelleme primer antikorlar, örneğin ms anti Histon ve rb anti nötrofil elastaz, sulandırınız. </li><li> Primer antikor bir damla üzerine tampon engelleme doğrudan nemli odasında fişleri kapağı ve 37 az 1 saat süreyle inkübe transfer ° C </li><li> PBS ile 3 kez 5 dakika yıkayınız. </li><li> Ikincil antikorlar, örneğin dk anti ms Cy2 ve tampon engelleme dk anti-rb Cy3 sulandırınız. </li><li> Sekonder antikoru bir damla üzerine nemli odasına fişleri kapağı ve 1 saat boyunca inkübe 37 aktarma ° C. </li><li> PBS ile 3 kez 5 dakika yıkayınız. Leke DNA UV eksitasyon veya Draq5 kadar kırmızı uyarma için ihtiyaç ve dist ile iki kez yıkamak Hoechst 33.342 (1 mcg / ml) ile 5 dakika ya, floresan mikroskopi seçenekleri bağlı. su. </li><li> Bir cam slayt üzerine Mowiol 20μl damla ayarlayın ve hücreleri ile kapak fişleri ters monte. Hücreleri kapak kayma ve slayt arasında olmak zorunda. Mowiol, açılan kapağı açılan kayma yerleştirilmiş homojen kalınlıkta ince bir tabaka oluşturacak. Normalde, örnek basın gerek yoktur. Olmayan daldırma lens kullanmak istiyorsanız, numunenin incelenmesi için hazır.Daldırma lensler ile mikroskobik inceleme için, Mowiol örnek kenarında katılaşmış kadar yaklaşık 1 saat için örnek kuru sağlar. </li></ol> 4. Hazırlanması NET&#39;ler Taramalı Elektron Mikroskobu (SEM) <ol><li> Hücreleri,% 2,5 glutaraldehid ile 24 plaka cam kapak bordroları düzeltmek Mesaj. </li><li> 24 kuyu kültür plaka hücreleri içeren cam kapak fişleri çıkartın ve bir damla su baş aşağı koymak. 3 kez 5 dakika boyunca bu gibi yıkayın. </li><li> 30 dakika süreyle% 0,5 OsO4 ve inkübe içeren 24-kuyu hücre kültürü plakası kapak fişleri geri aktarın. </li><li> 24 kuyu kültür plaka hücreleri içeren cam kapak fişleri çıkartın ve bir damla su baş aşağı koymak. 3 kez 5 dakika boyunca bu gibi yıkayın. </li><li> 30 dakika süreyle% 1 tannik asit ve inkübe içeren 24-kuyu hücre kültürü plakası kapak fişleri geri aktarın. </li><li> Adımları tekrarlayın 4,2-4,4 </li><li> Aşağıdaki rejimi (5 dk her) aracılığıyla dehydrate: <ul><li> % 30 etanol </li><li> % 50 etanol </li><li> % 70 etanol </li><li> % 80 etanol </li><li> % 90 etanol </li><li> % 100 etanol </li><li> % 100 etanol </li><li> % 100 etanol </li></ul></li><li> Kritik nokta kurutma makinesi ve kuru örnekler sığar kapağı aktarın. </li><li> Coat yüzey ince bir tabaka evaporatör kullanılarak 5nm platin / karbon tabaka ile örnek </li></ol> 5. Temsilcisi Sonuçlar: Izolasyon yöntem genellikle,% 95 daha yüksek bir saflığa sahip unstimulated yaşayabilir nötrofil verir. Stimülasyon sonrasında farklı zaman noktalarında sabit, NETosis düzleşme hücre, nükleer lobülleri kaybı, granül kaybı ve nükleer (yani histon) ve granül (yani artan bir örtüşme yol açar çekirdeği bütünlüğünü sırasında morfolojik değişiklikler immün protokol sırasını gösterir nötrofil elastaz) boyama. Bu protokol, nötrofil ile patojenlerin özel etkileşimlerini analiz etmek için bir başlangıç ​​noktası olarak hizmet verebilir. Bu etkileşim, Taramalı Elektron Mikroskobu için hazırlık protokolü kullanılarak daha ayrıntılı olarak disseke edilebilir. NET Floresans NET bileşenleri (mavi = DNA, kırmızı = histon, yeşil = nötrofil elastaz) lekeli uyarılan nötrofillerin örnek. NET lokalizasyonu, nükleer DNA ve histon yerelleştirme ve nötrofil elastaz için granül desen görüntüleri yanı sıra, göstermektedir. SEM PMA stimülasyon Olmayan uyarılır ve PMA-uyarılan nötrofillerin gösteren tarama elektron mikrografı. Stimülasyon sonra, nötrofil düzleştirmek ve NET&#39;ler üretmek. SEM NET&#39;ler ve Shigella NET&#39;ler sıkışıp Shigella bakterileri Yüksek çözünürlüklü SEM görüntü.

<ol>
	<li>Brinkmann, V. <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 .</li><li>Urban, C. F., Reichard, U., Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+capture+and+kill+Candida+albicans+yeast+and+hyphal+forms.">Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms.</a> Cell Microbiol. 8, 668-676 (2006).</li><li>M, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+amazonensis+promastigotes+induce+and+are+killed+by+neutrophil+extracellular+traps.">Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps.</a> PNAS. 106, 6748-6753 (2009).</li><li>Buchanan, J. T., Simpson, A. J., Aziz, R. K., Liu, G. Y., Kristian, S. A., Kotb, M., Feramisco, J., Nizet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNase+expression+allows+the+pathogen+group+A+Streptococcus+to+escape+killing+in+neutrophil+extracellular+traps.">DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps.</a> Curr Biol. 14, 396-400 (2006).</li><li>Fuchs, T. A. <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>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+suicide:+why+neutrophils+die+to+make+NETs.">Beneficial suicide: why neutrophils die to make NETs.</a> Nat Rev Microbiol. 5, 577-582 (2007).</li><li>Aga, E., Katschinski, D. M., van Zandbergen, G., Laufs, H., Hansen, B., Muller, K., Solbach, W., Laskay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+spontaneous+apoptosis+of+neutrophil+granulocytes+by+the+intracellular+parasite+Leishmania+major.">Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major.</a> J Immunol. , 169-898 (2002).</li></ol>

Sağlanan protokolü önemli saflık olmayan uyarılan nötrofillerin izolasyon, indüksiyon NET oluşumu ve NETosis sırasında morfolojik değişiklikleri analiz sağlayacaktır. Itina ile örneklerin tutarken, yani en gevşek bağlı NET&#39;ler kaybına neden olacak ağır yıkama koşullarına kaçınarak, farklı stimülasyon koşullar altında NET formasyonu (süresi, uyaran) miktarı mukayese edilebilir. Nötrofil / patojen etkileşimleri, uyaranlara dizisi, diğer bağışıklık hücreleri ile etkileşim: Bu bağlamda, verilen protokoller daha sofistike senaryolar analiz yöntemleri kurmak için bir başlangıç ​​noktası olarak hizmet verebilir.

neutrophil extracellular traps: how to generate and visualize them

Periferik kan lökosit nötrofil granülositler en bol bulunan bir grup. Profesyonel fagositler olarak, yutma bakteri ve hücre onları öldürmek phagosome ile antimikrobiyal granül sigorta. Biz nötrofil mikroorganizmaları öldüren ek bir yolu olduğunu bulundu: Etkinleştirme üzerine, birlikte ekstrasellüler lifler oluştururlar granül proteinleri ve kromatin serbest bağlama patojenler. Bu yeni yapılar, veya nötrofil Ekstrasellüler Tuzaklar (NET), virülans faktörleri aşağılamak ve bakteri 1, 2 ve parazitler 3 mantar öldürmek. NET&#39;ler yapısal omurga DNA ve DNases varlığı hızla bozulmuş. Böylece, DNases ifade bakteri 4 daha öldürücü. TEM, SEM, immünofloresan ve canlı hücre görüntüleme teknikleri birleştirerek bağıntılı mikroskopi kullanılarak stimülasyon üzerine, nötrofil çekirdekleri şekillerini kaybeder ve ab-ve heterokromatin homojenize gösterebilirim. Daha sonra, nükleer zarf ve granül membranlar NET bileşenleri karıştırma izin parçalanır. Son olarak, NET&#39;ler hücre zarı kesmeleri olarak yayımlanır. Bu hücre ölüm programı (NETosis) apoptoz ve nekroz farklıdır ve NADPH oksidaz 5 Reaktif Oksijen Türleri nesil bağlıdır. Nötrofil ekstrasellüler tuzakları akut inflamasyon bölgelerinde bol miktarda bulunmaktadır. NET&#39;ler bağlama mikroorganizmaların ki, yayılmasını önlemek ve virülans faktörleri düşürebilir ve böylece nötrofil hatta ömrü ötesinde antimikrobiyal fonksiyonu yerine getirmek için izin patojenleri öldürmek için yüksek antimikrobiyal ajanların yerel yoğunlaşmasını sağlamak, doğuştan gelen bağışıklık yanıtının bir formu olarak görünür. Kanıtlar artmaktadır, ancak bu NET&#39;ler olan hastalıklar da katılıyor oto-immün sendromlar kısırlık 6 aralığı. Biz, insan periferik kan 7 ila nötrofil Granülositler izole ve NET&#39;ler oluşturmak için onları teşvik etmek için yöntemleri açıklanmaktadır . Ayrıca ışık ve elektron mikroskobu NET&#39;ler görselleştirmek için protokolleri içerir.

Watch this Scientific Journal Video about Neutrophil Extracellular Traps: How to Generate and Visualize Them at JoVE.com

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Nötrofil Ekstrasellüler Tuzaklar (NET), patojen bakteriler, mantarlar ve parazitler mücadele için önemli bir doğuştan gelen bağışıklık mekanizması. Burada insan kan nötrofil granülositler izole etmek ve NET&#39;ler oluşturmak için bunları etkinleştirmek yöntemleri açıklanmaktadır. Biz ışık ve elektron mikroskobu NET&#39;ler görselleştirmek için hazırlama teknikleri mevcut.

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 ...

neutrophil-extracellular-traps-how-to-generate-and-visualize-them

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

86.3K Görüntüleme.  Max Planck Institute for Infection Biology. Nötrofil Ekstrasellüler Tuzaklar (NET), patojen bakteriler, mantarlar ve parazitler mücadele için önemli bir doğuştan gelen bağışıklık mekanizması. Burada insan kan nötrofil granülositler izole etmek ve NET'ler oluşturmak için bunları etkinleştirmek yöntemleri açıklanmaktadır. Biz ışık ve elektron mikroskobu NET'ler görselleştirmek için hazırlama teknikleri mevcut.

Video: Neutrophil Extracellular Traps: How to Generate and Visualize Them

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Tüp bebek Patojen Kaynaklı Nötrofil Trans-epitel Göçünü Değerlendirmek için Coculture Tahlili

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

İmmünoloji ve Enfeksiyon

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

İnsan nötrofil Akış odası Yapışma Deneyi

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

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 ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

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 ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Makrofaj hücre dışı tuzaklar Confocal mikroskobu kullanılarak görüntülenmesi

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

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. ...