1. Isolamento PMN dal sangue umano Utilizzare circa 24 ml di sangue umano con EDTA o eparina (10 U / ml) come anticoagulante. <ol><li> Aggiungere 6 ml HISTOPAQUE 1119 in una provetta da 15 ml Falcon e con attenzione strato di 5-7 ml di sangue intero in cima. </li><li> Centrifugare per 20 minuti a 800 xg senza frenare. </li><li> Aspirare e scartare giallastro e chiaro strato superiore e di trasferimento inferiore fase rossiccio contenenti granulociti in fresco tubi Falcon. </li><li> Lavare le cellule compilando i tubi Falcon con PBS e centrifugare per 10 minuti a 300 x g. </li><li> Nel frattempo preparare una soluzione Percoll al 100%, mescolando 18 ml Percoll con 2 ml di PBS 10x. Con 1x PBS preparare 4 soluzioni ml di 85%, 80%, 75%, 70% e 65% Percoll. </li><li> Preparare 2 tubi Falcon con un gradiente di Percoll stratificando 2 ml di ogni percentuale sopra l&#39;altro in ordine decrescente. </li><li> Dopo la centrifugazione eliminare il surnatante, combinare pellet e risospendere le cellule sedimentate in 4 ml di PBS. </li><li> Con attenzione il livello 2 ml di risospensione su ognuno dei gradienti. </li><li> Centrifugare per 20 minuti a 800 xg senza frenare. </li><li> Dopo la centrifugazione rimuovere strato superiore e la maggior parte del 65%-strato con PBMC e raccogliere bianco interfasi rimanente fino a 85%-strato in nuovi tubi Falcon. </li><li> Lavare le cellule riempiendo i tubi Falcon con PBS e centrifugare per 10 minuti a 300 x g. </li><li> Rimuovere le cellule sedimentate surnatante e risospendere (di solito&gt; 95% sono PMN) in 2 ml di PBS. </li><li> Contare le cellule utilizzando un emocitometro. </li></ol> 2. Attivazione di PMN <ol><li> Preparare un 24-pozzetti coltura cellulare mettendo una sterile 13 millimetri rotonda scivolare coperchio in vetro (# 1,5) in ciascun pozzetto </li><li> Semi di 2 x 10 5 cellule in 500μl RPMI (contenente 2% albumina sierica umana) per bene e incubare per 1h di CO 2 incubatore a 37 ° C. </li><li> Nel frattempo preparare una soluzione di 600 nM PMA in RPMI e aggiungere 100μl di cellule per pozzetto. Incubare da 15 minuti fino a 4 ore di CO 2 incubatore a 37 ° C. </li><li> Fissare le cellule nel 4% (concentrazione finale) paraformaldeide sciolto in PBS. </li></ol> PMA funge da controllo positivo e fino ad ora è l&#39;agente più potente per indurre la formazione di NET. In alternativa, altri stimoli o co-coltivazione con agenti patogeni possono essere utilizzati per l&#39;induzione NET. Lo stato della formazione rispettivi NET possono essere controllate, mentre il periodo di tempo sta procedendo, se ulteriori campioni paralleli sono preparati. Se un non-permeanti colorante DNA come Sytox verde (Invitrogen) si aggiunge alla non fisse celle, solo il DNA extracellulare verrà rilevato. Poiché la formazione di nuove reti è in qualche modo compromessa in presenza di Sytox, per ogni punto volta un campione parallelo deve essere utilizzato. 3. Rilevamento NET immunomarcatura NET sono molto fragili, anche dopo la fissazione e devono essere manipolati con grande cura, altrimenti la maggioranza si perde durante la preparazione. <ol><li> Rimuovere con attenzione scivola copertura in vetro con celle collegate dal 24-pozzetti cultura sollevandola al margine con un ago curvo e afferrare con una pinza sottile. Mettere il coperchio scivolare a testa in giù su una goccia di PBS. Questo può essere fatto su un foglio Parafilm che copre uno stand provetta. Lavare così 3 volte per 5 min. </li><li> Incubare i vetrini coprioggetto nello stesso modo in un calo dello 0,5% Triton X-100 per 1 min a RT al permeabilize cellule. Lavare 3 volte in PBS per 1 min. </li><li> Preparare una camera umida con Parafilm e un panno bagnato. Posare il coperchio scivola a testa in giù su una goccia di tampone di bloccaggio (5% asino siero) e incubare per 30 minuti a 37 ° C. </li><li> Diluire gli anticorpi primari, ad esempio ms contro Histon e rb contro elastasi neutrofila, nel tampone di bloccaggio. </li><li> Trasferimento di copertura scivola nella camera umida direttamente dal tampone di bloccaggio su una goccia di anticorpo primario e incubare per 1 ora a 37 ° C. </li><li> Lavare 3 volte per 5 minuti con PBS. </li><li> Diluire anticorpi secondari, ad esempio dk contro ms Cy2 e dk contro rb Cy3, nel tampone di bloccaggio. </li><li> Trasferimento coprire scivola nella camera umida su una goccia di anticorpo secondario e incubare per 1 ora a 37 ° C. </li><li> Lavare 3 volte per 5 minuti con PBS. A seconda delle opzioni di microscopia a fluorescenza, il DNA macchia per 5 minuti sia con Hoechst 33342 (1 mg / ml) che necessitano di eccitazione UV o con Draq5 per l&#39;eccitazione lontano rosso e lavare due volte con dist. acqua. </li><li> Impostare una goccia di 20μl Mowiol su un vetrino coprioggetto e montare con le cellule a testa in giù. Le cellule devono essere tra il vetrino e la diapositiva. La goccia di Mowiol formerà un sottile strato di spessore omogeneo in cui la polizza di copertura è posizionato sulla goccia. Normalmente, non c&#39;è bisogno di premere il campione. Se si desidera utilizzare non immersione lenti, il campione è pronto per l&#39;ispezione.Per l&#39;analisi microscopica con lenti ad immersione, lasciare che il campione secco per circa 1 ora fino a quando il Mowiol è solidificato al bordo del campione. </li></ol> 4. NET preparazione per microscopia elettronica a scansione (SEM) <ol><li> Messaggio fissare le cellule sul vetrino coprioggetto di vetro a 24-pozzetti con il 2,5% glutaraldeide. </li><li> Rimuovere i vetrini coprioggetto di vetro contenenti cellule dal 24-pozzetti cultura e messo a testa in giù su una goccia d&#39;acqua. Lavare così 3 volte per 5 min. </li><li> Trasferimento scivola coperchio posteriore in 24 pozzetti di coltura cellulare contenente 0,5% OsO4 e incubare per 30 min. </li><li> Rimuovere i vetrini coprioggetto di vetro contenenti cellule dal 24-pozzetti cultura e messo a testa in giù su una goccia d&#39;acqua. Lavare così 3 volte per 5 min. </li><li> Trasferimento scivola coperchio posteriore in 24 pozzetti di coltura cellulare contenente 1% di acido tannico e incubare per 30 min. </li><li> Ripetere i passaggi 4,2-4,4 </li><li> Disidratare attraverso il seguente schema posologico (5 minuti ciascuno): <ul><li> 30% di etanolo </li><li> Etanolo al 50% </li><li> Etanolo al 70% </li><li> 80% di etanolo </li><li> 90% di etanolo </li><li> 100% di etanolo </li><li> 100% di etanolo </li><li> 100% di etanolo </li></ul></li><li> Trasferimento di copertura scivola in asciugatrice punto critico e campioni secchi. </li><li> Superficie mano di campione con 5nm platino / carbonio con evaporatore strato sottile strato </li></ol> 5. Rappresentante dei risultati: Il metodo di isolamento produce di solito non stimolate neutrofili vitali con una purezza superiore al 95%. Quando fisso in diversi momenti dopo la stimolazione, il protocollo immunostaining mostra la sequenza di cambiamenti morfologici delle cellule durante NETosis appiattimento, la perdita di lobuli nucleare, la perdita di granulo e l&#39;integrità nucleo che porta ad un crescente sovrapposizione di nucleare (cioè gli istoni) e granulare (cioè neutrofila) colorazione. Questo protocollo può servire come punto di partenza per analizzare le interazioni degli agenti patogeni specifici con neutrofili. Questa interazione può essere sezionato in maggior dettaglio utilizzando il protocollo di preparazione per la microscopia elettronica a scansione. NET fluorescenza Esempio di neutrofili stimolati colorate per NET (blu = DNA, rosso = istone, verde = neutrofila). Le immagini mostrano, oltre alla localizzazione NET, la localizzazione nucleare del DNA e istoni e il modello granulare per l&#39;elastasi neutrofila. SEM PMA stimolazione Scansione al microscopio elettronico che mostra non neutrofili stimolati e PMA-stimolata. Dopo la stimolazione, i neutrofili appiattirsi e produrre NET. NET SEM e Shigella Maggiore risoluzione dell&#39;immagine SEM del batterio Shigella intrappolati nelle reti.

<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>

Il protocollo deve essere tale che l&#39;isolamento di non neutrofili stimolati a notevole purezza, l&#39;induzione della formazione del NET e l&#39;analisi dei cambiamenti morfologici durante NETosis. Quando si maneggiano i campioni con cura, evitando cioè dure condizioni di lavaggio che comporta la perdita di gran parte delle reti debolmente attaccato, la quantità di formazione NET in condizioni di stimolazione differenti (durata, stimolo) possono essere confrontati. A questo proposito, i protocolli previsti può servire come punto di partenza per stabilire i metodi per analizzare gli scenari più sofisticati: le interazioni neutrofili / patogeno, sequenza di stimoli, interazione con altre cellule immunitarie.

neutrophil extracellular traps: how to generate and visualize them

Granulociti neutrofili sono il gruppo più abbondante di leucociti nel sangue periferico. Come fagociti professionali, fagocitare i batteri e li uccidono intracellulare quando la loro miccia antimicrobica granuli con la phagosome. Abbiamo scoperto che i neutrofili sono un ulteriore modo di uccidere i microorganismi: al momento dell&#39;attivazione, rilasciano le proteine ​​dei granuli e cromatina che insieme formano le fibre extracellulari che legano gli agenti patogeni. Queste nuove strutture, o neutrofili Trappole extracellulare (NET), degradano fattori di virulenza e di uccidere i batteri 1, 2 e funghi parassiti 3. La spina dorsale strutturale del NET è il DNA, e sono rapidamente degradati in presenza di DNasi. Così, i batteri che esprimono DNasi sono più virulenti 4. Utilizzando la microscopia correlativa combinando TEM, SEM, immunofluorescenza e vivere tecniche di imaging cellulare, abbiamo potuto dimostrare che dopo la stimolazione, i nuclei di neutrofili perdono la loro forma e la eu-eterocromatina e omogeneizzare. Più tardi, l&#39;involucro nucleare e le membrane dei granuli disintegrano permettendo la miscelazione dei componenti NET. Infine, le reti sono rilasciati come rompe la membrana cellulare. Questo programma di morte cellulare (NETosis) è distinto da apoptosi e necrosi e dipende dalla generazione di specie reattive dell&#39;ossigeno da NADPH ossidasi 5. Neutrofili trappole extracellulare sono abbondanti nei siti di infiammazione acuta. NET sembrano essere una forma di risposta immunitaria innata che i microrganismi si legano, impediscono loro di diffondere e garantire una elevata concentrazione locale di agenti antimicrobici per degradare fattori di virulenza e uccidere gli agenti patogeni permettendo neutrofili per assolvere la loro funzione antimicrobica anche oltre la durata della loro vita. C&#39;è una crescente evidenza, tuttavia, che le reti sono anche coinvolti in malattie che vanno da auto-immune sindromi alla sterilità 6. Si descrivono i metodi per isolare i granulociti neutrofili dal sangue umano periferico 7 e stimolarli a formare NET. Inoltre abbiamo incluso i protocolli per visualizzare le reti in microscopia ottica ed elettronica.

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

Neutrofili Trappole extracellulare (NET) sono un importante meccanismo immunitario innato per combattere i batteri patogeni, funghi e parassiti. Qui si descrivono i metodi per isolare i granulociti neutrofili dal sangue umano e di attivare per formare NET. Vi presentiamo le tecniche di preparazione di visualizzare reti in microscopia ottica ed elettronica.

Neutrofili Trappole extracellulare: Come generare e poterle visualizzare

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

86.1K Views.  Max Planck Institute for Infection Biology. Neutrofili Trappole extracellulare (NET) sono un importante meccanismo immunitario innato per combattere i batteri patogeni, funghi e parassiti. Qui si descrivono i metodi per isolare i granulociti neutrofili dal sangue umano e di attivare per formare NET. Vi presentiamo le tecniche di preparazione di visualizzare reti in microscopia ottica ed elettronica.

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.

Semplificato umani neutrofili extracellulari Traps (NET) Isolamento e Manipolazione

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

Ricerca

Immunologia e infezioni

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.

Metodi per studiare lipidiche Alterazioni nei neutrofili e la formazione successiva di neutrofili extracellulare 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.

Visualizzazione del macrofago trappole extracellulari mediante microscopia 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.

Quantificazione automatizzata basata su immagini di trappole extracellulari neutrophil utilizzando 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'analisi di High-throughput per valutare e quantificare la trappola extracellulari del neutrofilo formazione

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.

Identificazione delle trappole extracellulari neutrophil in Paraffin-Embedded Feline Arterial Thrombi utilizzando la microscopia immunofluorescenza

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.

Analisi morfologica e composizionale di trappole extracellulari neutrofile indotte da stimoli microbici e chimici

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.

Quantificazione dei complessi mieloperossidasi-DNA e elastasi-DNA neutrofili da trappole extracellulari di neutrofili utilizzando un ELISA a sandwich modificato

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

Colorazione e imaging per visualizzare le trappole extracellulari dei neutrofili

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.

Autore in primo piano: Imaging di trappole extracellulari neutrofile in tessuti umani e di topo 

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.

Imaging in immunofluorescenza di trappole extracellulari di neutrofili in tessuti umani e murini

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