The authors would like to acknowledge Dr. Paul Kubes for his guidance and mentoring that were central in the construction of this work.

NOTE: All experiments were conducted in accordance with local institutional ethical guidelines.
1. Blood Drawing
<ol>
	<li>Through antecubital venipucture, collect 14 tubes of blood from a healthy volunteer into green top (heparinized) tubes and invert each tube before settling on ice. Collect blood within 10 - 15 min of experiment to ensure optimal neutrophil yield.</li>
</ol>
2. Neutrophil Isolation from Whole Blood
<ol>
	<li>Wash each green top tube of blood with 5 ml of PBS without Ca and Mg to dilute the blood. In each of 4 x 50 ml&#160;conical tubes, add 15 ml of Lymphocyte Separation Media (LSM) at RT. Using a 18 G needle mounted on a 60 ml&#160;syringe, carefully layer the diluted blood onto the LSM, creating a sharp LSM-blood interface. 
	NOTE: Avoid mixing of the layers as much as possible.</li>
	<li>Centrifuge at 800 x g for 30 min at 21 &#176;C without break. 
	NOTE: Sudden breaks can cause mixing of the different layers.</li>
	<li>Observe the erythrocytes and neutrophils sediment at the bottom. Aspirate and discard the top 2 layers (plasma and LSM) making sure to get rid of the interface between these layers which contains lymphocytes and mononuclear cells, leaving only the bottom red layer.</li>
	<li>To each tube, add 20 ml of Phosphate Buffer Saline (PBS) and 20 ml of 6% Dextran solution. Gently invert each tube to mix with the layer of erythrocytes and neutrophils and allow to sit at RT for 30 min. 
	NOTE: This will allow red blood cells (RBCs) to sediment at the bottom.</li>
	<li>After 30 min, transfer the neutrophil rich supernatant into fresh tubes and discard the RBC pellets. Centrifuge supernatants at 450 x g at 4 &#176;C for 5 min. After centrifugation, discard supernatant. The pellet will contain mostly neutrophils and few RBCs.</li>
	<li>Prepare a lysing solution by adding 0.5 ml of lysing buffer to 4.5 ml of sterile water. Lyse remaining RBC with the prepared 5 ml of lysing solution, transferring all resuspended pellets into one tube. Allow lysis at RT in the dark for 10 min.</li>
	<li>Centrifuge at 450 x g at 4 &#176;C for 5 min. Discard supernatant and wash pellet with 5 ml PBS without Ca and Mg and centrifuge again at 450 x g at 4 &#176;C to get rid of any remaining lysing solution. The pellet obtained at this point contains the neutrophils. Resuspend the pellet in 30 ml of cold 3% RPMI and put on ice. 
	NOTE: 3% RPMI is made by supplementing RPMI media with 3% Fetal Bovine Serum (FBS)</li>
	<li>Verify purity of the sample using Methylene blue staining. &#62;95% of cells should be granulocytes with multi-lobar nuclei. Use Trypan blue staining to verify &#62;95% viability of cells and count the final neutrophil yield. Dilute neutrophils in ice cold 3% RPMI to obtain a final concentration of 5 x 106 neutrophils/ml.</li>
</ol>
3. NETs Generation
<ol>
	<li>Stimulate neutrophils with 500 nM of PMA (per 30 ml of neutrophil solution) and incubate on a 150 x 25mm flat tissue culture dish with 20 mm grid for 4 hr at 37 &#176;C 5% CO2. This will allow NETosis.</li>
	<li>After 4 hr of stimulation, gently aspirate and discard the media, leaving the layer of NETs and neutrophils adhered at the bottom. Do not disrupt this layer.</li>
	<li>Using a total of 15 ml of cold PBS without Ca and Mg per dish, wash the bottom of each dish by pipetting 15 ml of PBS on the bottom of the dish in order to lift off all adherent material from the bottom.</li>
	<li>Collect solution obtained from washing each dish (step 3.3) in a 15 ml conical tube and centrifuge for 10 min at 450 x g at 4 &#176;C. Neutrophils and any remaining cells will pellet at the bottom, leaving a cell-free NET-rich supernatant.</li>
	<li>Divide supernatant into 1.5 ml micro-centrifuge tubes and spin for 10 min at 18,000 x g at 4 &#176;C. This will allow all DNA to pellet. 
	NOTE: Centrifugation in larger tubes is easier and less time consuming if such high speeds are attainable on the regular centrifuge, otherwise will need to divide supernatants in smaller tubes in order to use high speed micro-centrifuge.</li>
	<li>Discard supernatant and resuspend all pellets obtained together in ice cold PBS to a concentration corresponding to 2 x 107 neutrophils per 100 &#956;l of PBS. This will yield the cell-free NET stock that can be used for subsequent experiments.</li>
	<li>Measure DNA concentration in the sample obtained using spectrophotometry or alternate DNA quantification tool. An adequate concentration in the sample should range between 140 - 180 ng/&#956;l.</li>
</ol>
4. NET-Cancer Cell Static Adhesion Assay
<ol>
	<li>Add 100 &#956;l of the previously obtained NET stock per well in a 96 well flat bottom plate and allow to coat wells O/N at 4 &#176;C in the dark.</li>
	<li>Between 12 and 20 hr&#160;later, verify formation of a uniform monolayer of cell free NETs at the bottom of the wells under the microscope (Figure 1). At this point, gently aspirate all non-adherent material out of the wells, making sure not to disrupt the NET monolayer at the bottom.</li>
	<li>Add 100 &#956;l of 1% Bovine Serum Albumin (BSA) blocking solution to each well and leave for 1 h at RT.</li>
	<li>Detach A549 cancer cells from a T-75 flask of using 2 ml of 0.25% Trypsin solution. Once detached, add 10 ml of A549 media to trypsinized cells and centrifuge at 450 x g at 4 &#176;C for 5 min. Discard supernatant and resuspend cells in media to obtain a concentration of 2 x104 cancer cells per 100 &#956;l media. 
	NOTE: A549 cells were grown separately. Briefly, cells were cultured and maintained in DMEM F12 media containing 10% FBS and 1% Penicillin Streptomycin and incubated at 37 &#176;C 5% CO2. Once 70 - 80% cell confluence was reached, they were detached using 0.25% Trypsin-EDTA and resuspended in the same media described above.</li>
	<li>Stain cancer cells using CFSE by adding 1 &#956;l of CFSE per ml of media and leave to stain at RT for 10 min. After staining, centrifuge down at 450 x g at 4 &#176;C for 5 min then discard supernatant and resuspend cells in initial volume of media to obtain a concentration of 2 x104 cancer cells per 100 &#956;l media.</li>
	<li>After 1 hr of NETs blocking, gently aspirate blocking solution and add 2 x104 cancer cells in media, which is equivalent to 100 &#956;l, per well over the NET monolayer and allow to adhere for 90 min at 37 &#176;C 5% CO2. Gently aspirate the cells and add 100 &#956;l of PBS to each well to wash any non-adherent cells.</li>
	<li>In some wells, add 1000U of DNAse1 per well for 10 min before washing to degrade NETs. This will serve as negative control. In other wells, add 100 &#956;l of sterile water per well for 10 min, which will serve as the vehicle control (VC). 
	NOTE: 3 replicates per condition are usually performed to increase sample size.</li>
	<li>Aspirate and discard all solution in the wells leaving only NETs and adherent cancer cells at the bottom. Add 100 &#956;l of 4% formaldehyde solution per well to fix adherent cancer cells to NETS and read assay under the fluorescence microscope (Figure 1). Plot and analyze the results (Figure 3).</li>
</ol>

<ol>
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A., et al. <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 (2), 231-241 (2007).</li></ol>

The authors have nothing to disclose

The Neutrophil Extracellular Trap isolation protocol demonstrated in this video combines different techniques used in the literature for neutrophil isolation and NET formation. It simplifies a fairly complex and variable process and offers a very reliable and replicable way to isolate purified cell-free NETs with fewer steps than other protocols. Furthermore, readily available reagents are employed adding to the protocols simplicity and significantly reducing its cost. The application of NETs towards a static adhesion as...

Neutrophil extracellular traps (NETs) are newly discovered neutrophil-derived elements composed of strands of extracellular DNA decorated by hundreds of proteins and histones. They are secreted by neutrophils in the context of infection and inflammation following specific stimuli and they were initially recognized to be part of the neutrophil&#8217;s defense mechanisms against infections. They were first shown to promote trapping of various microbial invaders including bacteria, viruses and fungi 1-3. Trapping of the microbe would then lead to its destruction both directly by NET-associated proteins 3,4 and indirectly through local recruitment of phagocytic cells 5,6. The role of NETs however does not seem to be limited to host defense. More recently, they have been shown to play an important role in autoimmune diseases 7,8, thrombosis 9, pregnancy-related disorders 10 and even cancer progression 11,12. Accordingly, it appears that NETs play an important role in a large number of diverse physiologic processes. However, given the novel nature of such research, much remains to be elucidated with respect to the mechanisms by which NETs exert their effects. Herein, we present a simplified method for the isolation of NETs in the absence of neutrophils.
In the following video, we demonstrate a simplified and easy technique to isolate NETs from human whole blood. Cell-free NETs can then be used in a number of in vitro experiments including staining, imuunofluorescence, blotting as well as a number of assays such as adhesion, proliferation and migration. Other protocols exist 13, 19, however they are usually lengthy, complex, expensive, and often, low yield 13. This simplified protocol uses basic reagents and decreases the number of steps required to isolate neutrophils, therefore minimizing the length of the procedure while maximizing yield.
The following method combines different techniques for neutrophil isolation previously described in the literature to obtain a simple protocol yielding a very pure sample of live neutrophils. As suggested in the literature, venous blood is collected in EDTA tubes and used within 10 min to avoid neutrophil activation 14. We use Lymphocyte Separation Media (LSM) density gradient centrifugation to isolate granulocytes and red blood cells from heparinized whole blood, a method adapted from the Ficoll density centrifugation technique initially described by Boyum et al 15. LSM is a modification of the Boyum formulation that substitutes sodium diatrizoate for the sodium metrizoate and has been used successfully in many studies to isolate neutrophils 16-18.
Following differential density centrifugation, monocytes and lymphocytes are discarded; red blood cells are then sedimented using a 6% Dextran solution 14 and the remaining RBCs are lysed to obtain a population of pure neutrophils, which is verified with Trypan blue and Methylene blue staining.
Numerous agents have been used to induce NETosis both in vitro and in vivo, including lipopolysaccharide (LPS), and phorbol myristate acetate (PMA) and interleukins (IL-8) 3,5,6. In the following protocol, isolated neutrophils are stimulated with 500 nM PMA for 4 hr, which has been shown to be an adequate concentration to allow consistent and reliable NET formation without promoting apoptosis 19,20.
After isolation, we demonstrate how cell-free NETs obtained from this protocol can be used in an adhesion assay. DNAse1 is used to digest and degrade NETs as previously described and thus serves as a control 2,3,11. Other options include the use of Neutrophil elastase inhibitor (NEi) to inhibit NET formation, which is an acceptable alternative when the goal is to inhibit the NET formation rather than effect their degradation 11. Although NEi has a number of varied functions, it has been previously shown that NET deposition can be inhibited by NEi through blockage of chromatin decondensation, nuclear degranulation and neutrophil death 12

<table border="0" cellpadding="0" cellspacing="0" width="983">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:75px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:573px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Dulbecco&#8217;s Phosphate Buffered Saline (PBS), Modified, without calcium chloride and magnesium chloride</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">311-425-CL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lymphocyte Separation Medium (LSM)</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">305-010-CL</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextran hydrochloride (powder)</td>
			<td style="width:75px;">Spectrum</td>
			<td style="width:119px;">DE130</td>
			<td>6% Dextran solution is made by supplementing PBS with Ca and Mg with 6% Dextran powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RPMI-1640 Medium with L-glutamine</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">350-000-CL</td>
			<td>3% RPMI solution is made by supplementing RPMI with 3% Fetal Bovine serum</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BD Pharm Lyse Lysing buffer</td>
			<td style="width:75px;">BD Biosciences</td>
			<td style="width:119px;">555899</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phorbol 12-myristate 13-acetate (PMA)</td>
			<td style="width:75px;">Sigma-Alrdrich</td>
			<td style="width:119px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNAse1</td>
			<td>Roche</td>
			<td style="width:119px;">11284932001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F12 DMEM</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">319-075-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:75px;">Wisent</td>
			<td style="width:119px;">080-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:75px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CFSE</td>
			<td style="width:75px;">Invitrogen</td>
			<td style="width:119px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:75px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Integrid tissue culture dish with 20mm grid (150 x 25mm)</td>
			<td style="width:75px;">Falcon</td>
			<td style="width:119px;">353025</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Successful achievement of a static adhesion assay with NETs requires adequate coating of the wells with a monolayer of NETs before the addition of cancer cells (Figure 1). All subsequent steps must be done with care not to disrupt that layer. When an adequate layer of NETs is formed, it is expected to see significant cancer cells adhesion to NETs as seen in Figure 2A&#160;and 2C. This effect is abrogated by the addition of DNAse1, which degrades NETs as seen in F...

Watch this Scientific Journal Video about Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling at JoVE.com

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.

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 ...

simplified-human-neutrophil-extracellular-traps-nets-isolation

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Immunology and Infection

29.5K Views.  McGill University. 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.

Video: Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

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 ...

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. ...

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.

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

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

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

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.