1. Isolement PMN du sang humain Utilisez environ 24 ml de sang humain avec de l&#39;EDTA ou héparine (10 U / ml) comme anticoagulant. <ol><li> Ajouter 6 ml 1119 Histopaque à un tube Falcon de 15 ml et soigneusement ml de sang couche de 5 à 7 entières sur le dessus. </li><li> Centrifuger pendant 20 minutes à 800 xg sans freinage. </li><li> Aspirer et jeter jaunâtres et clairs couche supérieure et le transfert phase inférieure rougeâtre contenant des granulocytes dans des tubes Falcon frais. </li><li> Laver les cellules en remplissant des tubes Falcon avec du PBS et centrifuger pendant 10 minutes à 300 x g. </li><li> En attendant de préparer une solution de Percoll à 100% en mélangeant 18 ml de Percoll avec 2 ml de PBS 10x. Avec 1x PBS préparer 4 solutions ml de 85%, 80%, 75%, 70% et 65% de Percoll. </li><li> Préparer deux tubes Falcon avec un gradient de Percoll par superposition de 2 ml de chaque point de pourcentage au-dessus de l&#39;autre dans l&#39;ordre décroissant. </li><li> Après centrifugation éliminer le surnageant, mélanger et remettre granulés cellules sédimentées dans 4 ml de PBS. </li><li> Soigneusement couche 2 ml de la remise en suspension sur chacun des gradients. </li><li> Centrifuger pendant 20 minutes à 800 xg sans freinage. </li><li> Après centrifugation retirer la couche supérieure et la plupart des 65% de la couche avec des PBMC et de recueillir des blancs interphases restant jusqu&#39;à 85% de la couche dans des tubes Falcon de nouvelles. </li><li> Laver les cellules en remplissant des tubes Falcon avec du PBS et centrifuger pendant 10 minutes à 300 x g. </li><li> Retirer le surnageant et remettre les cellules sédimentées (généralement&gt; 95% sont PMN) dans 2ml de PBS. </li><li> Compter les cellules en utilisant un hématimètre. </li></ol> 2. Activation PMN <ol><li> Préparer une plaque de 24 puits de culture cellulaire en mettant une solution stérile de 13 mm de glissement rondes couvercle en verre (# 1,5) dans chaque puits </li><li> Graine 2 x 10 5 cellules dans 500μl RPMI (contenant 2% de sérum albumine humaine) par puits et incuber pendant 1h de CO 2 étuve à 37 ° C. </li><li> Pendant ce temps préparer une solution de 600 nM PMA dans RPMI et ajouter aux cellules 100 ul par puits. Incuber de 15 min à 4 h de CO 2 étuve à 37 ° C. </li><li> Fixer les cellules de 4% (concentration finale) paraformaldéhyde dissous dans du PBS. </li></ol> PMA sert de contrôle positif et est jusqu&#39;à présent l&#39;agent le plus puissant pour induire la formation de NET. Alternativement, d&#39;autres stimuli ou de co-culture avec des agents pathogènes peut être utilisé pour l&#39;induction NET. Le statut respectif de la formation des NET peut être vérifiée tout au cours du temps se déroule, si d&#39;autres échantillons sont préparés en parallèle. Si un colorant d&#39;ADN non-pénétrants, comme Sytox vert (Invitrogen) est ajouté aux cellules non-fixes, seul l&#39;ADN extracellulaire sera détectée. Depuis la formation de nouveaux filets est en quelque sorte altérée en présence d&#39;Sytox, pour chaque point dans le temps un échantillon parallèle doit être utilisé. 3. NET de détection par immunomarquage NET sont très fragiles, même après fixation et doivent être manipulés avec grand soin, sinon la majorité seront perdus lors de la préparation. <ol><li> Retirez délicatement lamelles de verre avec des cellules de la plaque attachée culture de 24 puits en le soulevant au bord d&#39;une aiguille courbe et la saisir avec une pince fine. Mettez la lamelle à l&#39;envers sur une goutte de PBS. Cela peut être fait sur une feuille de Parafilm couvrant un stand tube à essai. Laver comme celui-ci 3 fois pendant 5 min. </li><li> Incuber lamelles de la même manière à une baisse de 0,5% de Triton X-100 pendant 1 min à température ambiante à perméabiliser les cellules. Laver 3 fois en PBS pendant 1 min. </li><li> Préparer une chambre humide avec du parafilm et un tissu humide. Posez le couvercle glisse à l&#39;envers sur une goutte de tampon de blocage (5% âne sérum) et incuber pendant 30 min à 37 ° C. </li><li> Diluer anticorps primaires, par exemple la lutte contre Histon ms et RB anti-élastase neutrophile, en tampon de blocage. </li><li> Transfert couvercle glisse dans la chambre humide directement à partir du tampon de blocage sur une goutte d&#39;anticorps primaire et incuber pendant 1 h à 37 ° C. </li><li> Lavez 3 fois pendant 5 min avec du PBS. </li><li> Diluer anticorps secondaire, par exemple la lutte contre dk ms Cy2 et dk anti-rb Cy3, en tampon de blocage. </li><li> Transfert couvercle se glisse dans la chambre humide sur une goutte d&#39;anticorps secondaire et incuber pendant 1 h à 37 ° C. </li><li> Lavez 3 fois pendant 5 min avec du PBS. Selon vos options de microscopie par fluorescence, l&#39;ADN tache pendant 5 min, soit avec Hoechst 33342 (1 pg / ml), qui aura besoin d&#39;excitation UV ou Draq5 pour l&#39;excitation bien rouges et laver deux fois avec dist. de l&#39;eau. </li><li> Régler une baisse de Mowiol 20 pi sur une lame de verre et de monter des lamelles avec des cellules à l&#39;envers. Les cellules doivent être entre la lamelle et la lame. La baisse de Mowiol va former une couche mince d&#39;épaisseur homogène lorsque la lamelle est placée sur la goutte. Normalement, il n&#39;ya pas besoin d&#39;appuyer sur l&#39;échantillon. Si vous voulez utiliser des lentilles non-immersion, l&#39;échantillon est prêt pour l&#39;inspection.Pour l&#39;analyse microscopique avec des lentilles à immersion, laissez sécher le spécimen pendant environ 1 heure jusqu&#39;à ce que le Mowiol a solidifié au bord de l&#39;échantillon. </li></ol> 4. NET Préparation pour microscopie électronique à balayage (MEB) <ol><li> Message fixer les cellules sur les lamelles de verre de 24 puits plaque avec du glutaraldéhyde à 2,5%. </li><li> Retirer des lamelles de verre contenant des cellules de la plaque de culture à 24 puits et de mettre à l&#39;envers sur une goutte d&#39;eau. Laver comme celui-ci 3 fois pendant 5 min. </li><li> Transfert des lamelles de nouveau dans 24 puits, la plaque de culture cellulaire contenant 0,5% OsO4 et incuber pendant 30 min. </li><li> Retirer des lamelles de verre contenant des cellules de la plaque de culture à 24 puits et de mettre à l&#39;envers sur une goutte d&#39;eau. Laver comme celui-ci 3 fois pendant 5 min. </li><li> Transfert des lamelles de nouveau dans 24 puits, la plaque de culture cellulaire contenant 1% d&#39;acide tannique et incuber pendant 30 min. </li><li> Répétez les étapes 04/02 au 04/04 </li><li> Déshydrater à travers le schéma suivant (5min chacun): <ul><li> 30% d&#39;éthanol </li><li> 50% d&#39;éthanol </li><li> 70% d&#39;éthanol </li><li> 80% d&#39;éthanol </li><li> Éthanol à 90% </li><li> 100% d&#39;éthanol </li><li> 100% d&#39;éthanol </li><li> 100% d&#39;éthanol </li></ul></li><li> Transfert couvercle glisse dans sécheuse point critique et des échantillons secs. </li><li> Manteau de la surface de spécimen avec 5nm platine / carbone calque à l&#39;aide évaporateur à couche mince </li></ol> 5. Les résultats représentatifs: La méthode d&#39;isolement donne habituellement non stimulées neutrophiles viable avec une pureté supérieure à 95%. Lorsque fixé à différents moments après la stimulation, le protocole immunomarquage montre la séquence des changements morphologiques des cellules au cours NETosis aplatissement, une perte de lobules nucléaire, perte de granules et de l&#39;intégrité noyau qui aboutit à un chevauchement croissant de l&#39;énergie nucléaire (c&#39;est à dire des histones) et granulaires (ie élastase neutrophile) coloration. Ce protocole peut servir de point de départ pour analyser les interactions spécifiques des pathogènes avec des neutrophiles. Cette interaction peut être disséqué de façon plus détaillée en utilisant le protocole de préparation pour la microscopie électronique à balayage. NET fluorescence Exemple de neutrophiles stimulés colorées pour NET (bleu = ADN, rouge = histones, vert = élastase neutrophile). Les images montrent, outre la localisation NET, la localisation nucléaire de l&#39;ADN et les histones et le modèle granulaire pour l&#39;élastase neutrophile. SEM PMA de stimulation Micrographie électronique à balayage montrant non neutrophiles stimulés et stimulés par PMA. Après stimulation, les neutrophiles s&#39;aplatissent et produire NET. NET SEM et Shigella Une résolution plus élevée SEM image de bactéries Shigella piégés dans les filets.

<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>

Le protocole doit permettre à l&#39;isolement des non neutrophiles stimulés à la pureté considérable, l&#39;induction de la formation nette et l&#39;analyse des changements morphologiques au cours NETosis. Lors de la manipulation des échantillons avec soin, c&#39;est à dire en évitant les difficiles conditions de lavage qui se traduira par la perte de la plupart des filets lâchement, le montant de la formation des NET dans des conditions de stimulation différentes (durée, relance) peuvent être comparés. À cet égard, les protocoles prévus peut servir comme point de départ pour établir des méthodes pour analyser les scénarios plus sophistiqués: l&#39;interaction des neutrophiles / pathogène, la séquence de stimuli, interaction avec d&#39;autres cellules immunitaires.

neutrophil extracellular traps: how to generate and visualize them

Granulocytes neutrophiles sont le groupe le plus abondant de leucocytes dans le sang périphérique. Comme phagocytes professionnels, ils engloutissent les bactéries et les tuer quand leur niveau intracellulaire fusible antimicrobien granulés avec le phagosome. Nous avons constaté que les neutrophiles ont un moyen supplémentaire de tuer les microorganismes: lors de l&#39;activation, ils libèrent des protéines de granules et de la chromatine qui forment ensemble les fibres extracellulaires qui lient les agents pathogènes. Ces nouvelles structures, ou des neutrophiles pièges extracellulaires (EVF), dégradent les facteurs de virulence et de tuer les bactéries 1, 2 champignons et des parasites 3. L&#39;épine dorsale de structure des EVF est de l&#39;ADN, et ils sont rapidement dégradés en présence des DNases. Ainsi, les bactéries exprimant DNases sont plus virulents 4. En utilisant la microscopie corrélative alliant MET, MEB, immunofluorescence et de vivre des techniques d&#39;imagerie cellulaire, nous avons pu montrer que lors de la stimulation, les noyaux des neutrophiles perdent leur forme et de l&#39;UE et l&#39;hétérochromatine homogénéiser. Plus tard, l&#39;enveloppe nucléaire et les membranes des granules se désintègrent en permettant le mélange des composants NET. Enfin, les filets sont libérés que les pauses membrane cellulaire. Ce programme de mort cellulaire (NETosis) est distinct de l&#39;apoptose et nécrose et dépend de la génération d&#39;espèces réactives de l&#39;oxygène par la NADPH oxydase 5. Neutrophiles pièges extracellulaires sont abondants dans les sites d&#39;inflammation aiguë. NET semble être une forme de la réponse immunitaire innée que les microorganismes se lier, de les empêcher de se répandre, et assurer une forte concentration locale d&#39;agents antimicrobiens à dégrader les facteurs de virulence et de tuer les agents pathogènes permettant ainsi neutrophiles de remplir leur fonction antimicrobienne même au-delà de leur durée de vie. Il ya plus de preuves, cependant, que les filets sont également impliqués dans les maladies qui vont de syndromes auto-immuns à l&#39;infertilité 6. Nous décrivons des méthodes pour isoler granulocytes neutrophiles du sang périphérique humain 7 et les inciter à former des développements. Aussi nous inclure des protocoles de visualiser les filets dans la microscopie optique et électronique.

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

Neutrophiles pièges extracellulaires (NET) sont un important mécanisme immunitaire inné pour combattre les bactéries pathogènes, champignons et parasites. Nous décrivons ici les méthodes d&#39;isoler granulocytes neutrophiles dans le sang humain et de les activer pour former NET. Nous présentons les techniques de préparation de visualiser NET en microscopie optique et électronique.

Neutrophiles pièges extracellulaires: Comment générer et de les visualiser

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

86.0K vues.  Max Planck Institute for Infection Biology. Neutrophiles pièges extracellulaires (NET) sont un important mécanisme immunitaire inné pour combattre les bactéries pathogènes, champignons et parasites. Nous décrivons ici les méthodes d'isoler granulocytes neutrophiles dans le sang humain et de les activer pour former NET. Nous présentons les techniques de préparation de visualiser NET en microscopie optique et électronique.

Vidéo: 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.

Simplifié droits de neutrophiles extracellulaires Piège (NET) Isolement et Manipulation

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

Recherche

Immunologie et infection

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.

Méthodes d&#39;étude Les modifications lipidiques de neutrophiles et de la formation subséquente des neutrophiles pièges extracellulaires

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.

Visualisation des pièges extracellulaires de Macrophage à l’aide de la microscopie confocale

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.

Quantification automatisée basée sur l'image des pièges extracellulaires neutrophiles à l'aide de 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 test haut débit pour évaluer et quantifier les neutrophiles piège extracellulaire Formation

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.

Identification des pièges extracellulaires Neutrophil dans paraffin-Embedded Feline Arterial Thrombi à l’aide de la microscopie à l’immunofluorescence

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.

Analyse morphologique et compositionnelle des pièges extracellulaires neutrophiles induits par des stimuli microbiens et chimiques

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.

Quantification des complexes myéloperoxydase-ADN et neutrophile élastase-ADN à partir de pièges extracellulaires neutrophiles à l’aide d’un test ELISA sandwich modifié

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

Coloration et imagerie pour visualiser les pièges extracellulaires des neutrophiles

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.

Pleins feux sur l’auteur : Imagerie des pièges extracellulaires à neutrophiles dans les tissus humains et de souris 

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.

Imagerie par immunofluorescence des pièges extracellulaires des neutrophiles dans les tissus humains et murins

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