1. PMN Isolation from human blood
Use about 24 ml human blood with EDTA or Heparin (10 U/ml) as anticoagulant.
<ol>
 <li>Add 6 ml Histopaque 1119 to a 15 ml Falcon tube and carefully layer 5 to 7 ml whole blood on top.</li>
 <li>Centrifuge for 20 minutes at 800 x g without braking.</li>
 <li>Aspirate and discard yellowish and clear top layer and transfer lower reddish phase containing granulocytes into fresh Falcon tubes.</li>
 <li>Wash cells by filling up Falcon tubes with PBS and centrifuge for 10 minutes at 300 x g.</li>
 <li>In the meantime prepare a 100 % Percoll solution by mixing 18 ml Percoll with 2 ml 10x PBS. With 1x PBS prepare 4 ml solutions of 85 %, 80 %, 75 %, 70 % and 65 % Percoll.</li>
 <li>Prepare 2 Falcon tubes with a Percoll gradient by layering 2 ml of every percentage on top of each other in decreasing order.</li>
 <li>After centrifugation remove the supernatant, combine pellets and resuspend sedimented cells in 4 ml of PBS.</li>
 <li>Carefully layer 2 ml of the resuspension onto each of the gradients.</li>
 <li>Centrifuge for 20 minutes at 800 x g without braking.</li>
 <li>After centrifugation remove top layer and most of the 65%-layer with PBMCs and collect white remaining interphases until 85%-layer into new Falcon tubes.</li>
 <li>Wash cells by filling up the Falcon tubes with PBS and centrifuge for 10 minutes at 300 x g.</li>
 <li>Remove supernatant and resuspend sedimented cells (usually &gt;95% are PMN) in 2ml of PBS.</li>
 <li>Count cells using a hemocytometer.</li>
</ol>
2. Activating PMNs
<ol>
 <li>Prepare a 24-well cell culture plate by putting a sterile 13 mm round glass cover slip (# 1,5) into each well</li>
 <li>Seed 2 x 105 cells in 500&mu;l RPMI (containing 2 % human serum albumin) per well and incubate for 1h in CO2 incubator at 37&deg;C.</li>
 <li>Meanwhile prepare a 600 nM PMA solution in RPMI and add to cells 100&mu;l per well. Incubate from 15 min up to 4 h in CO2 incubator at 37&deg;C.</li>
 <li>Fix cells in 4% (end concentration) paraformaldehyde dissolved in PBS.</li>
</ol>
PMA serves as a positive control and is until now the most potent agent to induce NET formation. Alternatively, other stimuli or co-cultivation with pathogens can be used for NET induction.
The respective status of NET formation can be checked while the time course is proceeding, if additional parallel samples are prepared. If a non-permeant DNA dye like Sytox Green (Invitrogen) is added to the non-fixed cells, only extracellular DNA will be detected. Since formation of new NETs is somehow impaired in the presence of Sytox, for each time point one parallel sample has to be used.
3. NET detection by immunolabeling
NETs are very fragile even after fixation and have to be manipulated with great care, otherwise the majority will get lost during the preparation.
<ol>
 <li>Carefully remove glass cover slips with attached cells from 24-well culture plate by lifting it up at the edge with a curved needle and seize it with a fine forceps. Put the cover slip upside down on a drop of PBS. This can be done on a Parafilm sheet covering a test tube stand. Wash like this 3 times for 5 min. </li>
 <li>Incubate cover slips in the same manner in a drop of 0.5 % Triton X-100 for 1 min at RT to permeabilize cells. Wash 3 times in PBS for 1 min.</li>
 <li>Prepare a humid chamber with Parafilm and a wet tissue. Lay the cover slips upside down on a drop of blocking buffer (5% donkey serum) and incubate for 30 min at 37&deg;C.</li>
 <li>Dilute primary antibodies, e.g. ms anti Histon and rb anti Neutrophil Elastase, in blocking buffer.</li>
 <li>Transfer cover slips in the humid chamber directly from blocking buffer onto a drop of primary antibody and incubate for 1 h at 37&deg;C.</li>
 <li>Wash 3 times for 5 min with PBS.</li>
 <li>Dilute secondary antibodies, e.g. dk anti ms Cy2 and dk anti rb Cy3, in blocking buffer.</li>
 <li>Transfer cover slips into the humid chamber onto a drop of secondary antibody and incubate for 1 h at 37&deg;C.</li>
 <li>Wash 3 times for 5 min with PBS. Depending on your fluorescence microscopy options, stain DNA for 5 min either with Hoechst 33342 (1 &mu;g/ml) which will need UV excitation or with Draq5 for far red excitation and wash twice with dist. water.</li>
 <li>Set a 20&mu;l drop of Mowiol onto a glass slide and mount cover slips with cells upside down. The cells have to be between the cover slip and the slide. The drop of Mowiol will form a thin layer of homogenous thickness when the cover slip is positioned on the drop. Normally, there is no need to press the sample. If you want to use non-immersion lenses, the specimen is ready for inspection. For microscopic analysis with immersion lenses, let the specimen dry for about 1 hour until the Mowiol has solidified at the edge of the sample.</li>
</ol>
4. Preparing NETs for Scanning Electron Microscopy (SEM)
<ol>
 <li>Post fix cells on the glass cover slips in 24-well plate with 2,5 % glutaraldehyde.</li>
 <li>Remove glass cover slips containing cells from 24-well culture plate and put upside down on a drop of water. Wash like this 3 times for 5 min. </li>
 <li>Transfer cover slips back into 24-well cell culture plate containing 0,5% OsO4 and incubate for 30 min.</li>
 <li>Remove glass cover slips containing cells from 24-well culture plate and put upside down on a drop of water. Wash like this 3 times for 5 min. </li>
 <li>Transfer cover slips back into 24-well cell culture plate containing 1% tannic acid and incubate for 30 min.</li>
 <li>Repeat steps 4.2 to 4.4</li>
 <li>Dehydrate through the following regimen (5min each):
 <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 cover slips into critical point dryer and dry samples.</li>
 <li>Coat surface of specimen with 5nm platin/carbon layer using thin layer evaporator</li>
</ol>
5. Representative Results:
The isolation method usually yields unstimulated viable neutrophils with a purity greater than 95%. When fixed at different time points after stimulation, the immunostaining protocol shows the sequence of morphological changes during NETosis cell flattening, loss of nuclear lobules, loss of granule and nucleus integrity which leads to an increasing overlap of nuclear (i.e. histone) and granular (i.e. Neutrophil Elastase) staining. This protocol can serve as a starting point to analyze the specific interactions of pathogens with neutrophils. This interaction can be dissected in greater detail using the preparation protocol for Scanning Electron Microscopy.
NET Fluorescence
Sample of stimulated neutrophils stained for NET components (blue = DNA, red = histone, green = Neutrophil Elastase). The images show, besides the NET localization, the nuclear localization of DNA and histones and the granular pattern for Neutrophil Elastase.
SEM PMA stimulation
Scanning electron micrograph showing non stimulated and PMA-stimulated neutrophils. After stimulation, the neutrophils flatten out and produce NETs.
SEM NETs and Shigella
Higher resolution SEM image of Shigella bacteria trapped in NETs.

<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>

The provided protocol will allow the isolation of non stimulated neutrophils at considerable purity, the induction of NET formation and the analysis of morphological changes during NETosis. When handling the specimens with care, i.e. avoiding harsh washing conditions which will result in the loss of most of the loosely attached NETs, the amount of NET formation under different stimulation conditions (duration, stimulus) can be compared. In this respect, the provided protocols can serve as a starting point to establish methods to analyze more sophisticated scenarios: neutrophil/pathogen interactions, sequence of stimuli, interplay with other immune cells.

neutrophil extracellular traps: how to generate and visualize them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

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) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

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 Views.  Max Planck Institute for Infection Biology. Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

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.

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

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.

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.

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.

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.

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.

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.

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.

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

Staining and Imaging to Visualize Neutrophil Extracellular Traps

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.

Author Spotlight: Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues 

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.

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

Immunofluorescence Staining of Neutrophil Extracellular Traps in Tissue Samples

Begin by pipetting one drop of ready-to-use blocking solution with donkey serum onto the tissue samples. Then incubate the samples for 30 minutes at room temperature. Remove the excess blocking solution from the slides by tapping the edge against a hard surface.Dilute the primary antibodies in the antibody dilution buffer to prepare 100 microliters of the final solution for each sample. Set up the antibodies, one tube for each primary antibody and one for each isocontrol antibody. Evenly spread 100 microliters of isocontrol antibodies to one sample, and 100 microliters of myeloperoxidase and citrullinated histone H3, or myeloperoxidase and neutrophil elastase antibody dilution to the other sample.Place the slides in a wet chamber and store them overnight at four degrees Celsius. The following day, tap off excess primary antibody solution from the slides and stack them in a cuvette. Rinse the slides three times for five minutes each using Tris buffered saline with tween To remove the remaining primary antibody solution.Prepare two distinctly fluorescent secondary antibodies, donkey anti-goat for myeloperoxidase and donkey anti-rabbit for citrullinated histone H3 staining. Dilute each antibody to 7.5 micrograms per milliliter concentration in the antibody dilution buffer. Place the slides in a wet chamber and dispense 100 microliters of the secondary antibody solution onto each sample.Incubate them for 30 minutes at room temperature protected from light. Remove excess secondary antibody solution by tapping the slides. Put the slides in a slide rack.Submerge the slides three times for five minutes each in a staining jar filled with PBS to wash off unbound antibodies. Prepare the dappy solution and dilute it to a concentration of one microgram per milliliter with deionized water. Submerge the slide rack in a staining jar containing the dappy solution and incubate for five minutes at room temperature in the dark.Next, submerge the slide rack in a staining jar with PBS for five minutes to wash off any excess dappy solution. Finally, mount the samples using cover slips and mounting medium. The citrullinated histone H3 antibody only bound to the extracellular histones and showed no staining of intracellular histones.The human or mouse specific myeloperoxidase antibody showed no specific staining. While the universal human and mouse antibodies showed consistent good staining for myeloperoxidase. When no blocking agent was used, some mouse samples showed more non-specific and partial positive staining.When blocked, the five minutes blocking time showed good results compared to the 10 minutes blocking time. The higher temperatures in the microwave and water bath showed consistently moderate to good antigen retrieval. Whereas in a 60 degrees Celsius water bath there was only partially positive to no staining.For double staining, more favorable results were obtained for the samples incubated at 96 degrees Celsius water bath. However, exceeding the incubation time of 40 minutes at 96 degrees Celsius resulted in less intense antibody staining.