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

Research

JoVE Journal

Immunology and Infection

86.2K 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

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 ...

Imunologia e Infecção

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 ...

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 ...

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. ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

Identificação de Armadilhas Extracelulares De Neutrófilos em Trombo Arterial Felino Embutido parafina usando Microscopia de Imunofluorescência

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

Análise morfológica e composicional de armadilhas extracelulares de neutrófilos induzidas por estímulos microbianos e químicos

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

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.

Quantificação de complexos mieloperoxidase-DNA e elastase-DNA neutrofílico a partir de armadilhas extracelulares de neutrófilos usando um ELISA sanduíche modificado

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

Coloração e imagem para visualizar armadilhas extracelulares de neutrófilos

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 &#181;m thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.
Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

Autor em destaque: Imagens de armadilhas extracelulares de neutrófilos em tecidos humanos e de camundongos 

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

Imagem por Imunofluorescência de Armadilhas Extracelulares de Neutrófilos em Tecidos Humanos e de Camundongos

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...