1. PMN Isolation aus menschlichem Blut Verwenden Sie ca. 24 ml menschliches Blut mit EDTA oder Heparin (10 U / ml) als Antikoagulans. <ol><li> Add 6 ml Histopaque 1119 bis ein 15 ml Falcon-Röhrchen und sorgfältig Schicht von 5 bis 7 ml Vollblut an der Spitze. </li><li> Zentrifuge für 20 Minuten bei 800 xg ohne Bremse. </li><li> Absaugen und entsorgen gelblich und klar oberste Schicht und Transfer untere rötliche Phase mit Granulozyten in frischen Falcon-Röhrchen. </li><li> Wash-Zellen durch Auffüllen Falcon-Röhrchen mit PBS und Zentrifuge für 10 Minuten bei 300 x g. </li><li> In der Zwischenzeit bereiten eine 100% ige Percoll-Lösung durch Mischen 18 ml Percoll mit 2 ml 10x PBS. Mit 1x PBS vorzubereiten 4 ml Lösungen von 85%, 80%, 75%, 70% und 65% Percoll. </li><li> Bereiten Sie 2 Falcon Röhrchen mit einem Percoll-Gradienten durch die Schichtung von 2 ml jeder Prozentpunkt auf der jeweils anderen in absteigender Reihenfolge. </li><li> Nach der Zentrifugation den Überstand, kombinieren Pellets und resuspendieren sedimentierten Zellen in 4 ml PBS. </li><li> Sorgfältig Layer 2 ml der Resuspension auf jede der Steigungen. </li><li> Zentrifuge für 20 Minuten bei 800 xg ohne Bremse. </li><li> Nach der Zentrifugation zu entfernen oberste Schicht und die meisten der 65%-Schicht mit PBMCs und sammeln weißen verbleibenden Interphase bis 85%-Schicht in neue Falcon-Röhrchen. </li><li> Wash-Zellen durch Auffüllen der Falcon-Röhrchen mit PBS und Zentrifuge für 10 Minuten bei 300 x g. </li><li> Entfernen Überstand und resuspendieren sedimentierten Zellen (in der Regel&gt; 95% sind PMN) in 2 ml PBS. </li><li> Zählen von Zellen mit einer Zählkammer. </li></ol> 2. Aktivieren PMNs <ol><li> Bereiten Sie eine 24-well Zellkulturplatte, indem eine sterile 13 mm runden Deckglas (# 1,5) in jedes Well </li><li> Seed 2 x 10 5 Zellen in 500μl RPMI (mit 2% humanem Serum Albumin) pro Well und Inkubation für 1 Stunde in CO 2-Inkubator bei 37 ° C. </li><li> In der Zwischenzeit bereiten 600 nM PMA-Lösung in RPMI und fügen die Zellen 100 &amp;mgr; pro Vertiefung. Inkubieren von 15 min bis 4 h in CO 2-Inkubator bei 37 ° C. </li><li> Fix-Zellen in 4% (Endkonzentration) Paraformaldehyd in PBS gelöst. </li></ol> PMA dient als positive Kontrolle und ist bis heute die stärkste Agent NET zu induzieren. Alternativ können auch andere Reize oder die Co-Kultivierung mit Krankheitserregern für NET Induktion verwendet werden. Der jeweilige Status der NET-Bildung kann überprüft während der Zeit natürlich verläuft, wenn zusätzliche parallele Proben vorbereitet werden. Wenn ein Nicht-Permeationsmittel DNA-Farbstoff wie Sytox Green (Invitrogen), die nicht fixierten Zellen aufgenommen wird, werden nur extrazelluläre DNA nachgewiesen werden. Seit der Gründung von neuen NETs ist irgendwie in der Gegenwart von Sytox beeinträchtigt, für jeden Zeitpunkt eine parallele Probe verwendet werden. 3. NET-Erkennung durch Immunomarkierung NETs sind sehr zerbrechlich, auch nach der Fixierung und müssen mit großer Vorsicht behandelt werden, sonst wird die Mehrheit bei der Vorbereitung zu verlieren. <ol><li> Entfernen Sie vorsichtig Glasdeckgläschen mit anhaftenden Zellen aus 24-well Platte, indem Sie ihn bis an den Rand mit einer gebogenen Nadel und nutzen sie mit einer feinen Pinzette. Legen Sie das Deckglas umgekehrt auf einen Tropfen PBS. Dies kann auf einer Parafilm Blatt für ein Reagenzglas stand durchgeführt werden. Wash wie dieses 3-mal für 5 min. </li><li> Inkubieren Deckgläser auf die gleiche Weise in einem Rückgang von 0,5% Triton X-100 für 1 min bei RT Zellen permeabilisieren zu. Wash 3 mal in PBS für 1 min. </li><li> Bereiten Sie eine feuchte Kammer mit Parafilm und einem feuchten Tuch. Legen Sie das Deckgläschen kopfüber auf einen Tropfen Blockierungspuffer (5% Esel-Serum) und Inkubation für 30 min bei 37 ° C. </li><li> Verdünnen Primärantikörper, zB ms anti Histon und rb anti Neutrophile Elastase, in Blocking-Puffer. </li><li> Transfer Deckgläser in der feuchten Kammer direkt aus Blockierungspuffer auf einen Rückgang des primären Antikörpers und Inkubation für 1 h bei 37 ° C. </li><li> 3 x je 5 min mit PBS. </li><li> Verdünnen Sekundärantikörper, zB dk anti ms Cy2 und dk anti rb Cy3, in Blocking-Puffer. </li><li> Transfer Deckgläser in die feuchte Kammer auf einen Rückgang des sekundären Antikörper und Inkubation für 1 h bei 37 ° C. </li><li> 3 x je 5 min mit PBS. Abhängig von Ihrer Fluoreszenzmikroskopie Optionen Fleck DNA für 5 min entweder mit Hoechst 33342 (1 pg / ml), die UV-Anregung oder mit Draq5 für weit roten Anregung benötigen und waschen zweimal mit dest. Wasser. </li><li> Setzen Sie einen 20 &amp;mgr; l Tropfen Mowiol auf einen Objektträger und montieren Deckgläser mit Zellen auf den Kopf. Die Zellen müssen zwischen dem Deckglas und dem Objektträger werden. Die Tropfen Mowiol bilden eine dünne Schicht von homogenen Dicke, wenn das Deckglas auf den Tropfen positioniert ist. Normalerweise ist es nicht erforderlich, um die Probe zu drücken. Wenn Sie nicht Immersionslinsen verwenden möchten, wird die Probe zur Inspektion bereit.Für mikroskopische Analyse mit Immersionslinsen, lassen Sie die Probe trocken für etwa 1 Stunde, bis die Mowiol am Rand der Probe erstarrt ist. </li></ol> 4. Vorbereitung NETs für Rasterelektronenmikroskopie (SEM) <ol><li> Beitrag fix Zellen auf der Glasdeckgläschen in 24-Well-Platte mit 2,5% Glutaraldehyd. </li><li> Entfernen Glasdeckgläschen enthält Zellen, die aus 24-well Platten und legte den Kopf auf einen Tropfen Wasser. Wash wie dieses 3-mal für 5 min. </li><li> Transfer Deckgläschen wieder in 24-well Zellkulturplatte mit 0,5% OsO4 und Inkubation für 30 min. </li><li> Entfernen Glasdeckgläschen enthält Zellen, die aus 24-well Platten und legte den Kopf auf einen Tropfen Wasser. Wash wie dieses 3-mal für 5 min. </li><li> Transfer Deckgläschen wieder in 24-well Zellkulturplatte mit 1% Gerbsäure und Inkubation für 30 min. </li><li> Wiederholen Sie die Schritte 4,2 bis 4,4 </li><li> Entwässern durch die folgende Therapie (5min pro Stück): <ul><li> 30% Ethanol </li><li> 50% Ethanol </li><li> 70% Ethanol </li><li> 80% Ethanol </li><li> 90% Ethanol </li><li> 100% Ethanol </li><li> 100% Ethanol </li><li> 100% Ethanol </li></ul></li><li> Transfer Deckgläser in kritischen Punkt Trockner und trockenen Proben. </li><li> Coat Oberfläche der Probe mit 5 nm Platin / Kohlenstoff-Schicht mit Dünnschichtverdampfer </li></ol> 5. Repräsentative Ergebnisse: Die Isolierung Methode ergibt in der Regel nicht stimulierten lebensfähig Neutrophilen mit einer Reinheit von mehr als 95%. Wenn zu verschiedenen Zeitpunkten nach der Stimulation fixiert, zeigt die Immunfärbung Protokoll die Reihenfolge der morphologischen Veränderungen während NETosis Zelle Abflachung, Verlust der nuklearen Läppchen, Verlust von Granulat und Kern Integrität, die eine zunehmende Überschneidung der Kernenergie (dh Histon) und körnig (dh führt Neutrophile Elastase) Färbung. Dieses Protokoll kann als Ausgangspunkt, um die spezifischen Interaktionen von Pathogenen mit neutrophilen Granulozyten zu analysieren dienen. Diese Wechselwirkung kann in größerem Detail mit der Vorbereitung Protokoll für Scanning Electron Microscopy seziert werden. NET Fluorescence Beispiel der stimulierten Neutrophilen für NET-Komponenten (blau = DNA, rot = Histon, grün = Neutrophile Elastase) gefärbt. Die Bilder zeigen neben den NET-Lokalisierung, die nukleäre Lokalisation von DNA und Histonen und das körnige Muster für Neutrophile Elastase. SEM PMA-Stimulation Rasterelektronenmikroskopische Aufnahme zeigt nicht stimuliert und PMA-stimulierten neutrophilen Granulozyten. Nach Stimulation, glätten die Neutrophilen aus und produzieren NETs. SEM NETs und Shigella Höhere Auflösung REM-Aufnahme von Shigella Bakterien in NETs gefangen.

<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>

Die zur Verfügung gestellten Protokoll ermöglicht die Trennung von nicht stimulierten Neutrophilen auf erhebliche Reinheit, die Induktion von NET Bildung und die Analyse der morphologischen Veränderungen während NETosis. Beim Umgang mit den Proben mit Sorgfalt, dh rauen Wasch-Bedingungen, die in den Verlust des größten Teils der lose NETs Ergebnis zu vermeiden, kann die Menge des NET-Bildung unter verschiedenen Bedingungen Stimulation (Dauer, Stimulus) verglichen werden. Neutrophilen / Pathogen-Interaktionen, Folge von Reizen, Zusammenspiel mit anderen Immunzellen: In dieser Hinsicht kann das mitgelieferte Protokolle als Ausgangspunkt, um Methoden zu etablieren, um anspruchsvollere Szenarien analysieren dienen.

neutrophil extracellular traps: how to generate and visualize them

Neutrophile Granulozyten sind die häufigsten Gruppe von Leukozyten in das periphere Blut. Als professionelle Phagozyten, sie zu verschlingen Bakterien und töten sie intrazellulär, wenn ihre antimikrobielle Granula verschmelzen mit dem Phagosom. Wir fanden, dass Neutrophile eine zusätzliche Möglichkeit der Abtötung von Mikroorganismen haben: bei Aktivierung, geben sie Granulat Proteine ​​und Chromatin, die zusammen extrazelluläre Fasern binden Krankheitserreger. Diese neuartigen Strukturen oder Neutrophil Extracellular Traps (NETs), degradieren Virulenzfaktoren und töten Bakterien 1, 2 und Pilze Parasiten 3. Die strukturelle Rückgrat der NETs ist die DNA, und sie sind schnell abgebaut in Gegenwart von DNasen. So sind Bakterien exprimiert DNasen virulenter 4. Mit korrelative Mikroskopie kombiniert TEM, SEM, Immunfluoreszenz und Live Cell Imaging Verfahren konnten wir zeigen, dass nach Stimulation, die Kerne der Neutrophilen ihre Form verlieren und die EU-und Heterochromatin zu homogenisieren. Später zerfallen die Kernhülle und das Granulat Membranen ermöglicht das Mischen von NET-Komponenten. Schließlich werden die NETs als die Zellmembran Pausen freigegeben. Das Zelltod-Programm (NETosis) unterscheidet sich von Apoptose und Nekrose und hängt von der Generation von reaktiven Sauerstoffspezies durch NADPH-Oxidase-5. Neutrophile extrazelluläre Fallen sind reichlich an den Standorten der akuten Entzündung. NETs scheint eine Form der angeborenen Immunantwort sein, dass zu binden Mikroorganismen, Verhütung ihrer Ausbreitung, und sorgen für eine hohe lokale Konzentration von antimikrobiellen Wirkstoffen zu Virulenzfaktoren erniedrigen und töten Krankheitserreger so dass Neutrophile auf ihre antimikrobielle Funktion auch über ihre Lebensdauer zu erfüllen. Es gibt zunehmend Hinweise, jedoch, dass die NETs auch in Krankheiten, die beteiligten reichen von Autoimmunsyndrome zu Unfruchtbarkeit 6. Wir beschreiben Methoden zur neutrophilen Granulozyten aus peripherem menschlichem Blut 7 zu isolieren und sie dazu anregen NETs zu bilden. Auch wir sind Protokolle, um die Netze in Licht-und Elektronenmikroskopie zu visualisieren.

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

Neutrophil Extracellular Traps (NETs) sind ein wichtiger angeborenen Immunsystems Mechanismus auf pathogene Bakterien, Pilze und Parasiten zu kämpfen. Hier beschreiben wir Methoden, um neutrophilen Granulozyten aus menschlichem Blut zu isolieren und sie zu aktivieren, um NETs zu bilden. Wir präsentieren Vorbereitung Techniken, um NETs in Licht-und Elektronenmikroskopie zu visualisieren.

Neutrophil Extracellular Traps: So erzeugen und zu visualisieren

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

86.3K Aufrufe.  Max Planck Institute for Infection Biology. Neutrophil Extracellular Traps (NETs) sind ein wichtiger angeborenen Immunsystems Mechanismus auf pathogene Bakterien, Pilze und Parasiten zu kämpfen. Hier beschreiben wir Methoden, um neutrophilen Granulozyten aus menschlichem Blut zu isolieren und sie zu aktivieren, um NETs zu bilden. Wir präsentieren Vorbereitung Techniken, um NETs in Licht-und Elektronenmikroskopie zu visualisieren.

Serie: Neutrophil Extracellular Traps: How to Generate and Visualize Them

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Coculture Assay zur Bewertung pathogeninduzierter Neutrophiler Transepithelmigration

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

Forschung

Immunologie und Infektion

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Humanen neutrophilen Flusskammer Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

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.

Vereinfachte Menschen Neutrophil Extracellular Traps (NETs) Isolierung und Handhabung

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

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophilen-Isolierung und Analyse zur Bestimmung ihrer Rolle in der Lymphom-Zellempfindlichkeit therapeutische Mittel

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Methoden Lipidveränderungen in Neutrophilen und die anschließende Bildung von Neutrophilen Extracellular Traps zu studieren

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Eine einfache Fluoreszenztest zur Quantifizierung von Canine Neutrophil Extracellular Einklemmschutz

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualisierung von Makrophagen extrazelluläre fallen mit der konfokalen Mikroskopie

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In-vitro- Eckzahn Neutrophilenzahl extrazelluläre Trap Bildung: Dynamische und Quantitative Analyse von Fluoreszenz-Mikroskopie

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

Automatisierte bildbasierte Quantifizierung von neutrophilen extrazellulären Fallen mit 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.

Ein High-Throughput-Assay zu beurteilen und zu quantifizieren Neutrophilenzahl extrazelluläre Trap Bildung

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