1. PMN aislamiento de la sangre humana Utilice unos 24 ml de sangre humana con EDTA o heparina (10 U / ml) como anticoagulante. <ol><li> Agregar 6 ml Histopaque 1119 a un tubo Falcon de 15 ml y con mucho cuidado la capa de 5 a 7 ml de sangre entera en la parte superior. </li><li> Centrifugar durante 20 minutos a 800 xg sin frenar. </li><li> Aspirar y desechar la capa superior y amarillento claro y la transferencia de la fase inferior rojizo que contiene granulocitos en fresco tubos Falcon. </li><li> Lavar las células llenando tubos Falcon con PBS y centrifugar durante 10 minutos a 300 x g. </li><li> Mientras tanto preparar una solución de Percoll al 100% mediante la mezcla de 18 ml de Percoll con 2 ml de PBS 10x. Con 1x PBS preparar cuatro soluciones ml de 85%, 80%, 75%, 70% y 65% ​​de Percoll. </li><li> Preparar dos tubos Falcon con un gradiente de Percoll por capas de 2 ml de cada punto porcentual por encima de los demás en orden decreciente. </li><li> Después de la centrifugación separar el sobrenadante, se combinan pellets y resuspender las células sedimentadas en 4 ml de PBS. </li><li> Cuidado de la capa 2 ml de la resuspensión en cada uno de los gradientes. </li><li> Centrifugar durante 20 minutos a 800 xg sin frenar. </li><li> Después de la centrifugación quitar la capa superior y la mayoría de los 65% de la capa de PBMC y recoger blanco interfases restante hasta 85% en la capa de nuevos tubos Falcon. </li><li> Lavar las células llenando los tubos Falcon con PBS y centrifugar durante 10 minutos a 300 x g. </li><li> Eliminar las células sedimentadas sobrenadante y resuspender (normalmente&gt; 95% PMN) en 2 ml de PBS. </li><li> Recuento de células utilizando un hemocitómetro. </li></ol> 2. PMN activar <ol><li> Prepare una placa celular de 24 pocillos, poniendo una gasa estéril alrededor de 13 mm cubreobjetos de cristal (# 1,5) en cada pocillo </li><li> Semillas de 2 x 10 5 células en 500μl RPMI (que contiene 2% de albúmina sérica humana) por pocillo y se incuba durante 1 hora en CO 2 incubadora a 37 ° C. </li><li> Mientras tanto, preparar una solución de 600 nM PMA en RPMI y añadir a las células de 100μl por pocillo. Incubar de 15 min a 4 h de CO 2 incubadora a 37 ° C. </li><li> Fijar las células en el 4% (concentración final) paraformaldehido disuelto en PBS. </li></ol> PMA sirve como un control positivo y es hasta ahora el agente más potente para inducir la formación NET. Por otra parte, otros estímulos o co-cultivo con patógenos puede ser utilizado para la inducción de NET. Los estatutos respectivos de la formación de NET se puede comprobar mientras que la evolución en el tiempo es lineal, en caso adicional de muestras paralelas están preparados. Si un medio de contraste de ADN no permeables como Sytox Verde (Invitrogen) se añade a las células no-fijo, sólo el ADN extracelular será detectado. Desde la creación de nuevas redes es de alguna manera altera en presencia de Sytox, para cada momento una muestra paralela tiene que ser utilizado. 3. NET detección por inmunomarcación NET son muy frágiles, incluso después de la fijación y deben ser manipulados con mucho cuidado, de lo contrario la mayoría se pierden durante la preparación. <ol><li> Retire con cuidado el cristal de la cubierta se desliza con células adjunto de la placa de cultivo de 24 pocillos levantándolo en el borde con una aguja curva y tomar con una pinza fina. Coloque el cubreobjetos boca abajo sobre una gota de PBS. Esto se puede hacer en una hoja de Parafilm cubrir un puesto de tubo de ensayo. Lavado de la misma familia 3 veces por 5 min. </li><li> Incubar cubreobjetos de la misma manera en una caída del 0,5% Triton X-100 durante 1 minuto a temperatura ambiente para permeabilizar las células. Lavar 3 veces en PBS durante 1 min. </li><li> Preparar una cámara húmeda con Parafilm y un pañuelo de papel mojado. Ponga la tapa se desliza hacia abajo en una gota de tampón de bloqueo (5% burro suero) y se incuba durante 30 min a 37 ° C. </li><li> Diluir anticuerpos primarios, por ejemplo, ms contra Histon y rb contra la elastasa de los neutrófilos, en tampón de bloqueo. </li><li> La transferencia de la cubierta se desliza en la cámara húmeda directamente desde el amortiguador de bloqueo en una gota de anticuerpo primario y se incuba durante 1 hora a 37 ° C. </li><li> Lavar 3 veces durante 5 minutos con PBS. </li><li> Diluir secundaria de anticuerpos, por ejemplo, dk contra ms Cy2 y dk contra rb Cy3, en tampón de bloqueo. </li><li> La transferencia de la cubierta se desliza en la cámara húmeda en una gota de anticuerpo secundario y se incuba durante 1 hora a 37 ° C. </li><li> Lavar 3 veces durante 5 minutos con PBS. Dependiendo de las opciones de microscopía de fluorescencia, el ADN de la mancha durante 5 minutos, ya sea con Hoechst 33342 (1 mg / ml), que tendrá excitación UV o con Draq5 para la excitación del rojo lejano y lavar dos veces con dist. del agua. </li><li> Establecer una gota de 20μl Mowiol en un portaobjetos de vidrio y montaje de la cubierta se desliza con células de al revés. Las células tienen que ser entre el cubreobjetos y la corredera. La caída de Mowiol se forma una fina capa de espesor homogéneo cuando la hoja de la cubierta se coloca en la caída. Normalmente, no hay necesidad de presionar la muestra. Si desea utilizar lentes de no-inmersión, la muestra está lista para inspección.Para el análisis microscópico con lentes de inmersión, que la muestra se seque durante 1 hora hasta que la Mowiol ha solidificado en el borde de la muestra. </li></ol> 4. NET para la preparación de Microscopía Electrónica de Barrido (SEM) <ol><li> Después de fijar las células en las hojas de cubierta de vidrio de 24 y placa con 2,5% de glutaraldehído. </li><li> Eliminar hojas de vidrio cubierta con células de la placa de cultivo de 24 pocillos y poner boca abajo sobre una gota de agua. Lavado de la misma familia 3 veces por 5 min. </li><li> La transferencia de la cubierta se desliza de nuevo en 24 y placa de cultivo celular que contiene 0,5% OsO4 e incubar durante 30 min. </li><li> Eliminar hojas de vidrio cubierta con células de la placa de cultivo de 24 pocillos y poner boca abajo sobre una gota de agua. Lavado de la misma familia 3 veces por 5 min. </li><li> La transferencia de la cubierta se desliza de nuevo en 24 y placa de cultivo celular que contiene 1% de ácido tánico y se incuba durante 30 min. </li><li> Repita los pasos 4,2 hasta 4,4 </li><li> Deshidratan a través del siguiente régimen (5 minutos cada uno): <ul><li> 30% de etanol </li><li> 50% de etanol </li><li> 70% de etanol </li><li> 80% de etanol </li><li> 90% de etanol </li><li> 100% de etanol </li><li> 100% de etanol </li><li> 100% de etanol </li></ul></li><li> La transferencia de la cubierta se desliza en secador de punto crítico y muestras secas. </li><li> Capa superficial de la muestra con 5 nm platino / carbón de capa con evaporador de capa fina </li></ol> 5. Los resultados representativos: El método de aislamiento por lo general los rendimientos de los neutrófilos no estimulados viable con una pureza superior al 95%. Cuando se fija en diferentes puntos de tiempo después de la estimulación, el protocolo de inmunomarcación muestra la secuencia de cambios morfológicos en células NETosis aplanamiento, la pérdida de los lóbulos nucleares, la pérdida de gránulo y la integridad del núcleo que conduce a una creciente superposición de las armas nucleares (es decir, las histonas) y granular (es decir, elastasa de los neutrófilos) tinción. Este protocolo puede servir como punto de partida para analizar las interacciones específicas de los agentes patógenos con los neutrófilos. Esta interacción puede ser diseccionado con más detalle utilizando el protocolo de preparación para microscopía electrónica de barrido. NET de fluorescencia Muestra de neutrófilos estimulados teñidos para NET (azul = ADN, rojo = histonas, verde = elastasa de los neutrófilos). Las imágenes muestran, además de la localización NET, la localización nuclear de ADN y las histonas y el patrón granular para la elastasa de los neutrófilos. SEM PMA estimulación Micrografía electrónica de barrido que muestra no neutrófilos estimulados y PMA estimulada. Después de la estimulación, los neutrófilos se aplanan y producir NET. SEM NET y Shigella Mayor resolución de imagen SEM de la bacteria Shigella atrapados en las 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>

El protocolo deberá permitir que el aislamiento de los neutrófilos no estimulados con una pureza considerable, la inducción de la formación de NET y el análisis de los cambios morfológicos durante NETosis. Cuando se manipulan las muestras con cuidado, es decir, evitar las duras condiciones de lavado que se traducirá en la pérdida de la mayoría de las redes unen libremente, la cantidad de formación de NET en condiciones diferentes de estimulación (duración, estímulo) se pueden comparar. En este sentido, los protocolos que pueden servir como punto de partida para establecer los métodos para analizar los escenarios más sofisticados: las interacciones de neutrófilos / patógeno, la secuencia de estímulos, la interacción con otras células inmunes.

neutrophil extracellular traps: how to generate and visualize them

Granulocitos neutrófilos son el grupo más abundante de los leucocitos en la sangre periférica. Como fagocitos profesionales, las bacterias hundir y matar a intracelularmente cuando el fusible de gránulos de antimicrobianos con el fagosoma. Hemos encontrado que los neutrófilos tienen una forma adicional de matar a los microorganismos: la activación, liberan proteínas granulares y de la cromatina que en conjunto forman las fibras extracelulares que se unen los patógenos. Estas nuevas estructuras, o las trampas de neutrófilos extracelular (TNE), la degradación de los factores de virulencia y matar las bacterias 1, 2 hongos y parásitos 3. La columna vertebral estructural de la TNE es el ADN, y se degrada rápidamente en la presencia de DNasas. Por lo tanto, las bacterias que expresan DNasas son más virulentas 4. Utilizando microscopía correlativa combinar TEM, SEM, las técnicas de inmunofluorescencia de células y en directo de imágenes, podemos demostrar que la estimulación, los núcleos de los neutrófilos pierden su forma y homogeneizar la UE y la heterocromatina. Más tarde, la envoltura nuclear y las membranas de gránulos se desintegran permitiendo la mezcla de componentes NET. Por último, las redes se lanzan como los saltos de la membrana celular. Este programa de muerte celular (NETosis) es distinta de la apoptosis y la necrosis y depende de la generación de especies reactivas del oxígeno por la NADPH oxidasa 5. Neutrófilos trampas extracelulares son abundantes en los sitios de inflamación aguda. NET parece ser una forma de respuesta inmune innata que los microorganismos se unen, evitar que se propaguen, y asegurar una alta concentración local de agentes antimicrobianos para degradar los factores de virulencia y matar a los patógenos permitiendo así que los neutrófilos para cumplir con su función antimicrobiana incluso más allá de su vida. Cada vez hay más evidencia, sin embargo, que las redes también están implicados en enfermedades que van desde la auto-inmune síndromes de infertilidad 6. Se describen los métodos para aislar a los granulocitos neutrófilos de 7 de sangre periférica humana y estimular a formar NET. También se incluyen los protocolos para visualizar las redes en microscopía óptica y electrónica.

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

Las trampas de neutrófilos extracelular (TNE) es un importante mecanismo de inmunidad innata para luchar contra las bacterias patógenas, hongos y parásitos. A continuación se describen los métodos para aislar a los granulocitos neutrófilos de la sangre humana y para activarlos para formar NET. Se presentan las técnicas de preparación para visualizar NET en la microscopía óptica y electrónica.

Las trampas de neutrófilos extracelular: Cómo generar y visualizarlas

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 vistas.  Max Planck Institute for Infection Biology. Las trampas de neutrófilos extracelular (TNE) es un importante mecanismo de inmunidad innata para luchar contra las bacterias patógenas, hongos y parásitos. A continuación se describen los métodos para aislar a los granulocitos neutrófilos de la sangre humana y para activarlos para formar NET. Se presentan las técnicas de preparación para visualizar NET en la microscopía óptica y electrónica.

Video: 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 extracelular Trampas (TNE) Aislamiento y manipulación

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

Investigación

Inmunología e Infección

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 para el estudio de lípidos Las alteraciones en neutrófilos y la posterior formación de los neutrófilos extracelular Trampas

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.

Visualización de macrófagos trampas extracelular mediante Microscopía 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.

Cuantificación automatizada basada en imágenes de trampas extracelulares de neutrófilos mediante 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.

Un análisis de alto rendimiento para evaluar y cuantificar la formación de neutrófilos trampa extracelular

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.

Identificación de trampas extracelulares de neutrófilos en tromba arterial felino incrustada en parafina mediante microscopía de inmunofluorescencia

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.

Análisis morfológico y composicional de trampas extracelulares de neutrófilos inducidas por estímulos microbianos y 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.

Cuantificación de complejos mieloperoxidasa-ADN y neutrófilos elastasa-ADN a partir de trampas extracelulares de neutrófilos mediante el uso de un ELISA sándwich modificado

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

Tinción e imágenes para visualizar trampas 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 destacado: Imágenes de trampas extracelulares de neutrófilos en tejidos humanos y de ratón 

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.

Imágenes de inmunofluorescencia de trampas extracelulares de neutrófilos en tejidos humanos y de ratón

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