1. PMN de isolamento do sangue humano Use cerca de 24 ml de sangue humano com EDTA ou heparina (10 U / ml) como anticoagulante. <ol><li> Adicionar 6 ml Histopaque 1119 a um tubo de Falcon 15 ml e cuidadosamente camada ml de sangue 5-7 todo em cima. </li><li> Centrifugar por 20 minutos a 800 xg sem travões. </li><li> Aspirar e descartar amarelada e clara camada superior e inferior de transferência de fase avermelhada contendo granulócitos em tubos Falcon fresco. </li><li> Lave as células através do preenchimento de tubos Falcon com PBS e centrifugar por 10 minutos a 300 x g. </li><li> Nesse meio tempo preparar uma solução Percoll 100% através da mistura de 18 ml Percoll com 2 ml de PBS 10x. Com PBS 1x preparar quatro soluções ml de 85%, 80%, 75%, 70% e Percoll 65%. </li><li> Prepare 2 tubos Falcon com um gradiente de Percoll por camadas de 2 ml de cada porcentagem em cima uns dos outros em ordem decrescente. </li><li> Após a centrifugação remover o sobrenadante, combine pelotas e ressuspender as células sedimentadas em 4 ml de PBS. </li><li> Cuidadosamente camada 2 ml da ressuspensão em cada um dos gradientes. </li><li> Centrifugar por 20 minutos a 800 xg sem travões. </li><li> Após a centrifugação remover camada superior ea maioria dos 65% da camada de PBMCs e recolher com interfase branco restante até 85% da camada em tubos Falcon nova. </li><li> Lave as células por encher os tubos Falcon com PBS e centrifugar por 10 minutos a 300 x g. </li><li> Remover o sobrenadante e ressuspender as células sedimentadas (geralmente&gt; 95% são PMN) em 2 ml de PBS. </li><li> Contagem de células utilizando um hemocitômetro. </li></ol> 2. PMNs ativando <ol><li> Prepare uma placa de 24 poços de cultura de células, colocando uma estéril 13 milímetros rodada lamínula de vidro (# 1,5) em cada poço </li><li> Semente de 2 x 10 5 células em 500μl RPMI (contendo 2% de soro humano de albumina) por poço e incubar por 1h em incubadora de CO 2 a 37 ° C. </li><li> Enquanto isso, prepare a 600 nM solução PMA em RPMI e adicionar 100μl de células por poço. Incubar de 15 min até 4 h em incubadora de CO 2 a 37 ° C. </li><li> Fix células em 4% (concentração final) paraformaldeído dissolvido em PBS. </li></ol> PMA serve como um controle positivo e é até agora o agente mais potente para induzir a formação de NET. Alternativamente, outros estímulos ou co-cultivo com patógenos podem ser utilizados para a indução NET. Do respectivo estatuto de formação NET pode ser verificado quando o curso do tempo é de continuar, se forem complementares amostras paralelas estão preparados. Se um corante de DNA não-permeante como Sytox Green (Invitrogen) é adicionada às células não-fixa, apenas DNA extracelular será detectado. Desde a formação de novas redes é de alguma forma prejudicada na presença de Sytox, para cada ponto do tempo uma amostra paralela tem que ser usado. 3. NET detecção por imunomarcação NET são muito frágeis, mesmo após a fixação e tem que ser manipulado com muito cuidado, caso contrário, a maioria vai ficar perdido durante a preparação. <ol><li> Remova cuidadosamente lamínulas de vidro com células anexado a partir de 24 poços placa de cultura, levantando-a na borda com uma agulha curva e aproveitá-la com uma pinça fina. Coloque a lamínula de cabeça para baixo em uma gota de PBS. Isto pode ser feito em uma folha Parafilm cobrindo uma posição tubo de ensaio. Lavar 3 vezes como esta por 5 min. </li><li> Incubar lamínulas da mesma maneira em uma queda de 0,5% Triton X-100 por 1 min em temperatura ambiente para permeabilizar as células. Lavar 3 vezes em PBS por 1 min. </li><li> Prepare uma câmara úmida com Parafilm e um tecido molhado. Lay a tampa desliza de cabeça para baixo em uma gota de tampão de bloqueio (5% burro soro) e incubar por 30 min a 37 ° C. </li><li> Diluir anticorpos primários, por exemplo, ms anti Histon rb e anti elastase neutrofílica, em tampão de bloqueio. </li><li> Transferência de capa desliza na câmara úmida diretamente do tampão de bloqueio em uma gota de anticorpo primário e incubar por 1 hora a 37 ° C. </li><li> Lavar 3 vezes por 5 minutos com PBS. </li><li> Diluir anticorpos secundários, por exemplo dk anti ms Cy2 e dk anti rb Cy3, em tampão de bloqueio. </li><li> Transferência de capa desliza para dentro da câmara úmida em uma gota de anticorpo secundário e incubar por 1 hora a 37 ° C. </li><li> Lavar 3 vezes por 5 minutos com PBS. Dependendo das suas opções de microscopia de fluorescência, o DNA da mancha por 5 min ou com Hoechst 33342 (1 mg / ml), que vai precisar de excitação UV ou com DRAQ5 para excitação muito vermelho e lavar duas vezes com dist. água. </li><li> Definir uma gota de 20μl Mowiol numa lâmina de vidro e montar lamínulas com as células de cabeça para baixo. As células têm que ser entre a lamínula ea lâmina. A queda de Mowiol formará uma fina camada de espessura homogênea quando a lamínula é posicionado sobre a queda. Normalmente, não há necessidade de pressionar a amostra. Se você quiser usar lentes não-imersão, a amostra está pronta para a inspeção.Para análise microscópica com lentes de imersão, deixe a amostra secar por cerca de uma hora até que o Mowiol solidificou na borda da amostra. </li></ol> 4. NET preparando para Microscopia Eletrônica de Varredura (MEV) <ol><li> Pós corrigir células na tampa de vidro desliza em 24 poços da placa com glutaraldeído 2,5%. </li><li> Remova folhas tampa de vidro contendo células de 24 poços placa de cultura e colocar de cabeça para baixo em uma gota de água. Lavar 3 vezes como esta por 5 min. </li><li> Transferência de lamínulas de volta para 24 poços da placa de cultura de células contendo 0,5% OsO4 e incubar por 30 min. </li><li> Remova folhas tampa de vidro contendo células de 24 poços placa de cultura e colocar de cabeça para baixo em uma gota de água. Lavar 3 vezes como esta por 5 min. </li><li> Transferência de lamínulas de volta para 24 poços da placa de cultura de células contendo 1% de ácido tânico e incubar por 30 min. </li><li> Repita os passos de 4,2-4,4 </li><li> Desidratar através do seguinte esquema (5min cada): <ul><li> Etanol 30% </li><li> Etanol a 50% </li><li> Etanol 70% </li><li> Etanol 80% </li><li> Etanol 90% </li><li> Etanol 100% </li><li> Etanol 100% </li><li> Etanol 100% </li></ul></li><li> Transferência de capa desliza em secador de ponto crítico e amostras secas. </li><li> Superfície da camada de amostra com camada de platina / carbono 5nm usando evaporador camada fina </li></ol> 5. Resultados representativos: O método de isolamento geralmente produz unstimulated neutrófilos viável com uma pureza superior a 95%. Quando fixado em diferentes momentos após a estimulação, o protocolo imunocoloração mostra a seqüência de alterações morfológicas durante o celular NETosis achatamento, perda de lóbulos nuclear, perda de grânulos e integridade núcleo que conduz a uma crescente sobreposição de armas nucleares (ou seja, histona) e granular (ie A elastase neutrofílica coloração). Este protocolo pode servir como ponto de partida para analisar as interações específicas de patógenos com neutrófilos. Essa interação pode ser dissecada em maior detalhe utilizando o protocolo de preparação para microscopia eletrônica de varredura. Fluorescência NET Amostra de neutrófilos estimulados manchada para componentes de NET (azul = DNA, histona vermelho = verde = elastase neutrofílica). As imagens mostram, além da localização NET, a localização nuclear do DNA e as histonas eo padrão granular para a elastase neutrofílica. SEM estimulação PMA Micrografia eletrônica de varredura mostrando neutrófilos não estimulados e PMA-estimulado. Após a estimulação, os neutrófilos achatar e produzir NETs. SEM NET e Shigella Maior resolução de imagem SEM de bactérias Shigella presos em redes.

<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>

O protocolo permitirá que desde o isolamento de neutrófilos não estimulados a pureza considerável, a indução da formação de NET e da análise das alterações morfológicas durante NETosis. Ao manusear as amostras com cuidado, ou seja, evitando duras condições de lavagem, que resultará na perda da maioria dos NETs frouxamente ligados, a quantidade de formação NET em condições diferentes de estimulação (duração, de estímulo) podem ser comparados. A este respeito, os protocolos fornecidos podem servir como ponto de partida para estabelecer métodos para analisar cenários mais sofisticados: interações de neutrófilos / patógeno, a seqüência de estímulos, interação com outras células do sistema imunológico.

neutrophil extracellular traps: how to generate and visualize them

Granulócitos neutrófilos são o grupo mais abundante de leucócitos no sangue periférico. Como fagócitos profissionais, eles bactérias engolir e matá-los intracelularmente quando seu fusível grânulos antimicrobiana com o fagossomo. Descobrimos que os neutrófilos têm uma maneira adicional de matar microorganismos: após a ativação, liberam proteínas granulares e cromatina que juntos formam as fibras extracelulares que se ligam patógenos. Estas novas estruturas, ou neutrófilos Traps Extracelular (NET), degradam fatores de virulência e matar as bactérias 1, fungos e parasitas 2 3. A espinha dorsal estrutural da NET é o DNA, e eles são rapidamente degradados na presença de DNases. Assim, as bactérias expressando DNases são mais virulentas 4. Usando a microscopia correlativos combinando TEM, SEM, técnicas de imagem de imunofluorescência e de células vivas, poderíamos mostrar que com a estimulação, os núcleos de neutrófilos perdem sua forma e eu e heterocromatina homogeneizar. Mais tarde, o envelope nuclear e as membranas grânulos desintegrar permitindo a mistura de componentes NET. Finalmente, as redes são lançadas como as quebras de membrana celular. Este programa de morte celular (NETosis) é distinta da apoptose e necrose e depende da geração de espécies reativas de oxigênio pela NADPH oxidase 5. Neutrófilos armadilhas extracelular são abundantes nos locais de inflamação aguda. NET parecem ser uma forma de resposta imune inata que os microorganismos se ligam, evitar a sua disseminação, e garantir uma alta concentração local de agentes antimicrobianos para degradar fatores de virulência e matar agentes patogênicos, permitindo assim neutrófilos para cumprir sua função antimicrobiana, mesmo para além do seu tempo de vida. Há crescentes evidências, no entanto, que as redes também estão envolvidos em doenças que vão desde auto-imune síndromes à infertilidade 6. Descrevemos métodos para isolar Granulócitos neutrófilos de sangue periférico humano 7 e estimulá-los a formar redes. Também incluímos protocolos para visualizar a NET em microscopia óptica e eletrônica.

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

Neutrófilos Traps Extracelular (NET) são um mecanismo inato importante imunológico para combater as bactérias patogênicas, fungos e parasitas. Aqui nós descrevemos métodos para isolar granulócitos neutrófilos do sangue humano e para ativá-las para formar redes. Nós apresentamos técnicas de preparação para visualizar NETs em microscopia óptica e eletrônica.

Neutrófilos Traps Extracelular: Como Gerar e visualizá-los

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

86.3K visualizações.  Max Planck Institute for Infection Biology. Neutrófilos Traps Extracelular (NET) são um mecanismo inato importante imunológico para combater as bactérias patogênicas, fungos e parasitas. Aqui nós descrevemos métodos para isolar granulócitos neutrófilos do sangue humano e para ativá-las para formar redes. Nós apresentamos técnicas de preparação para visualizar NETs em microscopia óptica e eletrônica.

Vídeo: 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.

In vitro Ensaio de cocultura para avaliar migração trans epitelial de neutrófilo induzida por patógenos

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

Imunologia e Infecção

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.

Fluxo de Neutrófilos Humanos Secção Ensaio de Adesão

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.

Simplificado Humanos neutrófilos extracelulares Traps (NETs) Isolamento e Manuseio

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.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

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.

Métodos de estudo de lipídios Alterações nos neutrófilos ea subsequente formação de neutrófilos Extracelular Traps

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.

A fluorescência ensaio simples para a quantificação de Canine neutrófilos Extracelular Armadilha lançamento

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.

Visualizando o macrófago armadilhas extracelular usando microscopia Confocal

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.

In Vitro Formação canina neutrófilos extracelular armadilha: Análise dinâmica e quantitativa por microscopia de fluorescência

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.

Quantificação automatizada baseada em imagem de armadilhas extracelulares de neutrófilos usando NETQUANT

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.

Um ensaio de alta produtividade para avaliar e quantificar a formação de neutrófilos extracelular armadilha

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