Name: Neutrophil Extracellular Traps: How to Generate and Visualize Them (Video) | JoVE Journal
Uploaded: 2010-02-24T17:00:00.000+00:00
Duration: 14 min 17 s
Description: Learn to isolate neutrophil granulocytes from human blood and stimulate NET formation using chemical or biological stimuli. Visualize neutrophil extracellular traps by immunofluorescence and scanning electron microscopy.

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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/pubmed?term=((Neutrophil+extracellular+traps+kill+bacteria%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neutrophil extracellular traps kill bacteria.</a> Science. 303, 1532-1535 Forthcoming.</li>
<li>Urban, C. F., Reichard, U., Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+capture+and+kill+Candida+albicans+yeast+and+hyphal+forms%5BTitle%5D)+AND+%22Cell+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Leishmania+amazonensis+promastigotes+induce+and+are+killed+by+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">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/pubmed?term=((DNase+expression+allows+the+pathogen+group+A+Streptococcus+to+escape+killing+in+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">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/pubmed?term=((Novel+cell+death+program+leads+to+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Beneficial+suicide%3A+why+neutrophils+die+to+make+NETs%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Inhibition+of+the+spontaneous+apoptosis+of+neutrophil+granulocytes+by+the+intracellular+parasite+Leishmania+major%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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 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.

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 or nets are a recently identified mechanism for neutrophil mediated clearance of pathogens by releasing a combination of cytoplasmic and granule proteins, as well as chromatin neutrophils form extracellular fibers that trap pathogens. In this video, granulocytes are isolated from human whole blood by histopath density gradient centrifugation. Neutrophils are isolated from the granulocyte preparation by per coral gradient.Centrifugation seeded onto cover glass and stimulated to form nets using chemical or biological stimuli, the cells are then processed for immuno labeling or em for visualization of nets. Hi, I'm Christian Gosman from the microscopy core facility at the Max Plunk Institute for Infection Biology. Today we will show you a procedure to purify neutrophil granulocytes from human blood and stimulate them to generate neutrophil extracellular traps or nets.We will then visualize nets by immunofluorescence and scanning electron microscopy. We use this procedure in our lab to study the mechanisms that lead to net formation and to analyze the antimicrobial properties of nets. So let's get started.Begin this protocol in a lamina flow hood by preparing six milliliters of histo 1119 into each of four to 15 milliliter Falcon tubes. Carefully layer five to seven milliliters of whole blood over the histo in each tube centrifuge tubes for 20 minutes at about 800 times G, ensuring that the break is off once the centrifugation has completed. Carefully remove the tubes from the centrifuge.The tube will contain four clearly separated layers from top to bottom. These are a dark red layer containing erythrocytes. A light red layer containing the neutrophil granulocytes with some erythrocytes, a whiteish yellow layer containing leukocytes and serum, and a clear yellow layer of serum.Again, working in the lamina flow hood. Use the perpet attached to a vacuum system to carefully aspirate the top yellow layer. Then using a pipette transfer the reddish phase containing granulocytes from each tube into a new falcon tube.Next, fill the tubes with PBS to wash the granulocytes. Then pellet them by centrifugation for 10 minutes at about 300 times G while the cells are spinning. Prepare a 100%per coll solution by mixing 18 milliliters of per co with two milliliters of 10 times PBS.Then use the 100%per coll solution and one times PBS to prepare four milliliters each of 65 70, 75, 80, and 85%per call. Use these solutions to prepare per core gradients by carefully layering two milliliters of each solution into each of two tubes. Begin by petting two milliliters of the 85%solution into each tube.Then on top of the 85%per coll, carefully layer two milliliters of the 80%per co. Continue layering two milliliters of each solution in decreasing order such that the 65%solution is layered on top. Remove the sedimented leukocytes from the centrifuge under the hood.Discard the supinate and resuspend the cells in one milliliter of PBS. Then combine the resuspended cells in a fresh tube. Carefully layer two milliliters of the resuspended granulocytes onto each of the perol gradients.Then centrifuge for 20 minutes at about 800 times G, ensuring that the break is off after centrifugation. The interfaces should be clearly visible due to the highest cell density. Discard the top layer and most of the 65%layer, which contain peripheral blood mononuclear cells or p BMCs.Collect the 65%70%interface together with a 70%75%and 80%85%Interface from each gradient and transfer them to new fresh falcon tubes. Wash the cells by filling the tube with PBS and centrifuge for 10 minutes at about 300 times G to pellet the cells after centrifugation. Remove the supinate and resuspend the sedimented, polymorph and nuclear leukocytes or PMNs in two milliliters of PBS.At this point, 95%or more of the cells in the preparation will be mns. Using a hemo cytometer determines cell counts, then see 200, 000 cells in 500 microliters of RPMI onto sterile round cover slips placed in culture plates with 15 millimeter size wells. Incubate for one hour in a 5%CO2 incubator at 37 degrees Celsius to allow the cells to attach to the cover slips after one hour.Add 100 microliters of RPMI containing the desired stimulant. For a time course experiment, add the stimulant sequentially, starting with the longest stimulation time for each conditional time point. Be sure to prepare a positive control by adding 600 nano molars of PMA in RPMI.Also, for each condition or time point, prepare a negative control by adding RPMI alone incubate for up to four hours in a CO2 incubator at 37 degrees Celsius during stimulation, the formation of nets can be monitored using bright field microscopy. Shortly after stimulation, nuclei changed their form dramatically. They lose their LOEs round up, then expand at the desired time point at 600 microliters of room temperature, 8%paraform aldehyde solution to each well.To bring the final concentration to 4%incubate for two to four hours for immuno labeling. Place a paraform sheet on top of a test tube rack with three times eight bore holes of approximately 15 millimeter diameter. To form wells, fill wells with PBS.Use a curved needle to lift up each of the cover slips from the paraldehyde on the culture plate. Then using forceps, grasp the cover slip and invert it onto the surface of a drop of PBS on the paraform. At this point, neutrophil extracellular traps are very fragile and great care should be taken during manipulation to avoid cell loss.After five minutes, repeat the wash by transferring the cover slips to new drops of PBS. Repeat for a total of three washes. Next, transfer the cover slips to a drop of room temperature 0.5%Triton X 100 for one minute to permeate the cells.Then wash three times in PBS for one minute each following the wash. Proceed with the staining according to the accompanying written protocol. Performing each of the incubations and wash steps by inverting the cover glass on the paraform.Once the cells have been stained, place a 20 microliter drop of MO onto a glass slide and mount the cover slips face down with the cells between the cover slip and the slide. After one hour, the MOY should have solidified and the cells are ready for microscopic analysis. With immersion lenses, prepare the cells for scanning electron microscopy under a chemical fume hood.Post fixx the cells on the glass cover slips in a 24 well plate with 2.5%glutaraldehyde in PBS for 30 minutes. During the incubation, place a paraform sheet on top of a test tube. Stand with three times eight bore holes of approximately 15 millimeter diameter.To form wells. Fill the wells with distilled water. Use a curved needle to lift up each of the cover slips from the glutaraldehyde solution.Then using forceps, grasp the cover slip and transfer it to a drop of distilled water on the paraform wash by transferring the cover slips to drops of distilled water on Paraform. Repeat for a total of three washes, five minutes each transfer cover slips back into the wells of the cell culture plate containing 0.5%osmium tetroxide in distilled water and incubate for 30 minutes at room temperature. Remove the cover slips from the plate and wash three times in distilled water as before.Then return them to the 24 well plate with 0.5 milliliters of 1%tannic acid in each well and incubate for 30 minutes. Next, wash the cover slips three times five minutes each. Then repeat the fixation with osmium tetroxide and the wash.Next, cover the test tube. Stand with a new piece of paraform. Dehydrate the cells through a series of incubations in the following ethanol solutions for five minutes each 30, 50, 70, 80, 9100, 100.And finally, one more time in 100%ethanol. Submerge the rack in an ethanol filled specimen boat. Quickly place the cover slips into the metal rack, making sure that the specimens do not dry out.Then transfer the specimen boat into a cooled critical point dryer or CPD. Fill the chamber of the CPD with a liquid CO2. Leave the inlet valve fully open to maintain liquid level and as soon as a phase of ethanol is visible under the CO2 phase on the bottom of the chamber, open the drain valve at the bottom.To remove most of the ethanol. Allow the chamber to flush for about 30 minutes. After flushing, close the inlet valve and lower the liquid level to just below the top of the boat.Run hot water through the jacket of the CPD. When the temperature reaches 36 to 38 degrees Celsius and the pressure rises to 1, 200 pounds per square inch, the liquid gas boundary line will disappear and the specimens will be above critical point. Once the critical point has been reached, vent the gas off slowly over a five minute period.To avoid condensation, the temperature should remain around 40 degrees Celsius. During this time, open the door and remove the boat. Confirm that the dehydrating solvent has evaporated.Stick the cover slips right side up to the adhesive on the aluminum specimen holder and transfer them into the high resolution coating system. Evacuate the coating cylinder to two times 10 to the minus five millibars. Then set the instrument to 1.8 kilovolts and 70 milliamps, and monitor the thickness of the platon carbon layer with a quartz crystal thickness until the layer reaches five nanometers.Vent the coating cylinder of the high resolution coating system. Then using forceps, remove the samples from the coter. Bring the chamber of the SEM to normal pressure.Open the door. Then insert the samples into the specimen holder and evacuate the chamber. For SEM analysis.When fixed at different time, points after stimulation, morphological changes during NETosis can be observed by immunofluorescence microscopy. In this figure, the DNA is shown in blue histon. DNA complex is shown in red and neutrophil elastase.A granule marker is shown in green during the first phase of NETosis, only the nu nuclear periphery in condensed nuclei. Later, when the chromatin is more decon condensed, most of the nucleus is stained in the decon condensed nets. Staining is homogenous.This panel of images details the overlap of nuclear and granular staining that occurs with NETosis. This scanning electron micrograph shows non stimulated neutrophils after stimulation with Ella bacterium, the neutrophils flatten out and produce neutrophil extracellular traps.Traps. Here a gel bacterium has been trapped in nets.Bacteria such as gel are immobilized and killed by the enzymes present in nets. We've just shown you how to isolate neutrophil granulocytes from human blood and to stimulate them in order to induce formation of neutrophil extracellular traps. These can then be further analyzed by immunofluorescence or scanning electron microscopy.When doing this procedure, it is important to remember that nets are very fragile even after fixation. In order to minimize loss of nets, we take the cover slips with the stimulated neutrophils from the well cavities and do most of the procedure on the surface of buffer drops on. Even then, it's important to move the specimens gently and slowly.So that's it. Thanks for watching and good luck with your experiments.

neutrophil-extracellular-traps-how-to-generate-and-visualize-them

Research

JoVE Journal

Immunology and Infection

This video demonstrates the fluorescent staining of neutrophil extracellular traps (NETs), the extracellular DNA components expelled by neutrophils. This technique enables the visualization of adhesive interaction between cancer cells and NETs.

Fluorescence Microscopy of Neutrophil Extracellular Traps or NETs: A Technique to Observe Adhesive Interaction Between Cancer Cells and NETs

This video demonstrates an in vitro technique to generate and isolate neutrophil extracellular traps (NETs). Neutrophils are isolated from peripheral blood and stimulated with phorbol myristate acetate (PMA) to produce NETs. During incubation, the neutrophils undergo a process of cell death (NETosis) to release NETs comprising decondensed chromatin mixed with granular cytoplasmic proteins.

An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

This video demonstrates the induction and visualization of neutrophil extracellular traps or NETs in pre-labeled neutrophils using patient serum containing anti-neutrophil cytoplasmic antibodies. Furthermore, using an impermeable DNA dye and fluorescence microscopy enables the visualization of NETs.

Induction and Visualization of Neutrophil Extracellular Traps in Pre-Labeled Neutrophils

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.

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

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.

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

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.

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

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.

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

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.

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Learn to isolate neutrophil granulocytes from human blood and stimulate NET formation using chemical or biological stimuli. Visualize neutrophil extracellular traps by immunofluorescence and scanning electron microscopy.

Neutrophil Extracellular Traps: How to Generate and Visualize Them

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