The corresponding author was funded by the Morris Animal Foundation (D15CA-907). The study was supported by funds from the University of California, Davis, Center for Equine Health and Center for Companion Animal Health (2016-24-F).&#160;The authors would like to acknowledge Geena Ng for her assistance with the figures and Nghi Nguyen for her assistance with the video.

All methods described here were approved by the Institutional Animal Care and Use Committee at the University of California, Davis (protocol number: 18338).
1. Blood Collection
<ol>
	<li>Draw 10 mL of blood from either the cephalic or jugular vein using a 21 G needle by syringe aspiration.</li>
	<li>To avoid excessive shear stress, remove the needle from the syringe before transferring blood into tube(s) containing sodium heparin (75 USP). Gently invert the tubes a few times to ensure adequate mixing of anticoagulant and blood. Check for evidence of clots or red cell aggregates before placing the samples on ice.</li>
</ol>
2. Neutrophil Isolation
<ol>
	<li>Dilute anticoagulated blood with an equal volume of ice-cold Dubecco&#8217;s phosphate-buffered saline (DPBS) (pH 7.4, without divalent cations). Transfer 8 mL of diluted blood into a 50 mL polypropylene conical tube containing 25 mL of 3% dextran (From Leuconostoc spp. dissolved in 0.9% sodium chloride solution). Place the tube upright in room temperature for 30 min to allow aggregation and sedimentation of erythrocytes.</li>
	<li>Carefully layer 5 mL of leukocyte-rich plasma (Figure 1) on top of 5 mL of density gradient media in a 14 mL polypropylene round-bottom tube. Be careful not to mix the 2 solutions11.</li>
	<li>Centrifuge the tubes at 400 x g with acceleration/deceleration (A/D) set to 0 (no brake) for 30 min at room temperature.</li>
	<li>Using a 5 mL serological pipet collect the granulocyte cell layer by bypassing the plasma and peripheral blood mononuclear cell (PBMC) layers (Figure 1). Use a new pipet each time to minimize contamination.</li>
	<li>Place 4 to 6 mL of the polymorphnuclear (PMN) cell layer in a 50 mL polypropylene conical tube. Since a small amount of erythrocyte aggregates may be aspirated along with the PMN layer, use a 1 mL serological pipet to gently remove the erythrocyte aggregates that are settled at the bottom of the tube.</li>
	<li>Lyse the remaining erythrocytes with 20 mL of cold ultrapure water. Gently invert the tube multiple times for 30 to 60 s to ensure adequate lysing before adding an equal volume of ice-cold 1.8% sodium chloride solution.</li>
	<li>Centrifuge at 112 x g for 10 min at 4 &#176;C, A/D set to 0.</li>
	<li>Repeat the erythrocyte lysis step if this pellet appears red. Discard the supernatant carefully with a serological pipet and gently resuspend the pellet in 100 &#181;L of DPBS (5 mM dextrose, 0.9 mM MgCl2, 1.9 mM CaCl2 and 1% bovine serum albumin, pH 7.3). Combine all resuspended cells and place on ice.</li>
	<li>Perform a cell count using an automated cell analyzer and verify the count by a hemocytometer. If necessary, concentrate PMNs onto a glass slide using a cytocentrifuge (1,000 rpm, 5 min) and determine the percentage of neutrophils by counting the number of neutrophils out of the total number of cells.</li>
	<li>Determine the viability of the neutrophils by performing a trypan blue exclusion test as outlined by Strober et al.12&#160;Successful isolation should yield a viability from 95 to 100% and neutrophils should be greater than 95%.</li>
	<li>Determine the concentration of neutrophils by multiplying the total cell count with the percent viability and percent neutrophils. Successful isolation should yield a concentration of neutrophils from 1.5 to 6 x 106/mL.</li>
	<li>Dilute neutrophils to a final concentration of 1 x 106/mL with DPBS containing 5 mM dextrose, 0.9 mM MgCl2, 1.9 mM CaCl2, and 1% bovine serum albumin with pH adjusted to 7.3.</li>
</ol>
3. Fluorescent Microscopy of Live Neutrophils
<ol>
	<li>Place 200 to 400 &#956;L of neutrophils (1 x 106/mL) into each well of a poly-L-lysine coated 12-well culture plate. Allow neutrophils to adhere to the bottom of the wells by incubating the cells at 37 &#176;C for 30 min.</li>
	<li>Label nucleic acids with 1 &#956;M of one of two nucleic acid dyes, one that stains in cells with intact membranes and one that stains in cells with permeable membranes (see Table of Materials), for 10 min at room temperature.</li>
	<li>Activate neutrophils with 100 &#956;g/mL lipopolysaccharide (LPS) (E. coli O55.B5), 100 nM phorbol 12-myristate 13-acetate (PMA) as positive control, or equivalent volume of DPBS as negative control for 180 min at 37 &#176;C.</li>
	<li>Acquire images using the GFP (Excitation: 470/22 nm, Emission: 525/50 nm) and Texas Red channels (Excitation: 585/29 nm, Emission: 628/32 nm) on a fluorescence microscopy at 40X magnification at the following time points: 30, 90, 120, and 180 min.</li>
</ol>
4. NET Quantification and Detection of NET Components Using Immunofluorescence
<ol>
	<li>Carefully place 18 mm diameter Poly-D-lysine coated cover slips into the wells of a 12-well culture plate. Label the wells appropriately.</li>
	<li>Seed 100 to 200 &#956;L of isolated neutrophils (1 x 106/mL) directly onto the coverslips and allow neutrophils to adhere to the cover slips by incubating the cells at 37 &#176;C for 30 min.</li>
	<li>Activate neutrophils as described in 3.3. Inhibit NET formation by pretreating neutrophils with 200 &#181;&#924; Cl-amidine (15 min, 37 &#176;C) prior to activation as previously described by Li et al.13 Ensure that the proper vehicle control (DMSO) is included in the experiment.</li>
	<li>Carefully remove cover slips with fine forceps. Layer a paraffin film sheet over a test tube stand to create shallow wells and place 2 to 3 drops of 4% PFA in each well10. Label the wells appropriately and gently lay the cover slips upside down onto the PFA-filled wells. Allow cells to be fixed at room temperature for 20 min.</li>
	<li>Wash the cells 3 times in DPBS for 5 min, by moving them from one well to the next.</li>
	<li>If immunolabelling cannot be performed shortly after neutrophil activation and fixation, allow cover slips to be air dried by placing the cover slips face-up on a piece of absorbance paper. Cover slips can be stored for up to 1 week face-up in a labelled 12-well culture plate at 4 &#176;C until further analysis. Wash the cells 3 times in DPBS for 1 min using the same technique as described in 4.4 before proceeding to the next step.</li>
	<li>To permeabilize cells, gently lay the cover slip upside down on a drop of 1% NP-40 for 1 min using the technique described in 4.4. Wash 3 times in DPBS for 1 min on a rocker.</li>
	<li>Randomly assign each sample to a number and use a diamond point marker to label the coverslips accordingly.</li>
	<li>Prepare and label individual Petri dishes lined with paraffin film and place 100 to 200 &#956;L of 5% goat serum onto the film (blocking buffer). Lay the coverslips upside down on the blocking buffer. Place on a rocker and incubate for 1 h at 37 &#176;C.</li>
	<li>Wash 3 times in DPBS for 5 min on a rocker.</li>
	<li>Dilute the primary antibody (1:400 rabbit polyclonal anti-citrullinated histone H3 (citH3) in 5% goat serum). Incubate cells in labeled Petri dishes lined with paraffin film. Lay each cover slip upside down on 50 to 100 &#956;L of the diluted antibody solution. Place on a rocker and incubate for 1 h at 37&#176;C.</li>
	<li>Wash 3 times in DPBS for 5 min on a rocker.</li>
	<li>Dilute secondary antibody conjugated to a fluorophore (1:200) in 5% goat serum. Incubate cells in labeled Petri dishes lined with paraffin film. Lay each cover slip upside down on 50 to 100 &#956;L of the diluted antibody solution. Place on a rocker and incubate 1 h at 37 &#176;C in the dark.</li>
	<li>Wash 3 times in DPBS for 5 min on a rocker in the dark.</li>
	<li>For concurrent detection of myeloperoxidase (MPO) follow the following steps for a modified double immunolabeling protocol as described by Li et al.13</li>
	<li>Incubate cells in labeled Petri dishes lined with paraffin film. Lay each cover slip upside down on 100 to 200 &#956;L of 10% rabbit serum under gentle rocking (overnight, 4 &#176;C, in the dark).</li>
	<li>Wash 3 times in DPBS for 5 min on a rocker in the dark.</li>
	<li>Incubate cells in 50 &#956;g/mL unconjugated goat anti-rabbit Fab fragments for 2 h at room temperature on a rocker in the dark.</li>
	<li>Wash 3 times in DPBS for 5 min on a rocker in the dark.</li>
	<li>Dilute primary antibody (1:200 rabbit polyclonal anti-human MPO antibody) in 5% goat serum. Incubate cells in labeled Petri dishes lined with paraffin film. Lay each cover slip upside down on 50 to 100 &#956;L of the diluted antibody solution. Place on a rocker and incubate 1 h at 37&#176;C in the dark.</li>
	<li>Dilute secondary antibody conjugated to a fluorophore in 5% goat serum and incubate as described in 4.8</li>
	<li>Wash 3 times in DPBS for 5 min on a rocker in the dark.</li>
	<li>Interference controls should be prepared by excluding incubation with either primary antibodies in the second immune-labelling step.</li>
	<li>Stain DNA with 300 nM of 4&#8217;,6-Diamidion-2-Phenylindole, Dihydrochloride (DAPI) for 5 min in the dark at room temperature.</li>
	<li>Wash 3 times in DPBS for 1 min on a rocker in the dark.</li>
	<li>Apply a drop (~50 &#956;l) of antifade mountant onto a glass slide. Gently tap the edge of the coverslip on an absorbent paper to remove excess buffer before mounting the coverslip with the cells upside down. The mounting medium should form a thin layer between the coverslip and the glass slide. To remove air bubbles, very gently press on the coverslip with fine forceps.</li>
	<li>Allow samples to cure overnight in the dark at 4 &#176;C. The mounting medium should harden for microscopic analysis with immersion lenses. Store samples in 4 &#176;C in the dark.</li>
</ol>
5. Neutrophil Extracellular Trap Quantification
<ol>
	<li>Blind the operator(s) acquiring and analyzing the microscope images to the experimental conditions.</li>
	<li>Using a fluorescence microscope at 40X magnification, take 10 images randomly from different regions of each coverslip in an experiment under the respective channels. Ensure that the exposure time, brightness and contrast of each channel are kept consistent throughout the acquisition of images.</li>
	<li>Analyze the images using available software such as ImageJ (NIH). Identify NETs based on co-localization of cfDNA, citH3 and MPO (Figure 3A, B). Quantify the number of NETs and neutrophils in 10 random fields for each experimental condition. Numbers of NETs released can be expressed as a ratio (number of NETs: total number of neutrophils) for each experimental condition.</li>
	<li>To evaluate the treatment effects on intracellular citH3 expression, count the number of neutrophils with intracellular citH3 and number of neutrophils without intracellular citH3 in 10 random fields at 40X magnification under the respective channels (Figure 3, red arrows). Intracellular citH3 expression can be expressed as the ratio of number of neutrophils with intracellular citH3 to the number of neutrophils without intracellular citH3.</li>
</ol>

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	<li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nature Reviews Immunology. 6 (3), 173-182 (2006).</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> Nature Reviews Microbiology. 5 (8), 577-582 (2007).</li><li>Kaplan, M. 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The authors have nothing to disclose.

We present here a protocol to observe the changes in nuclei conformation and cfDNA release in living canine neutrophils using both a cell permeant dye and a cell impermeant dye. The main advantage of this assay is that it allows for real-time detection of NET formation by high-resolution microscopy in live neutrophils without cell fixation, therefore, providing a simple and valuable tool for observing in vitro NET formation. Since this assay does not require antibodies for the detection of NET components, it is ...

Neutrophils are short-lived granulocytes responsible for the initial defense against invading pathogens. Neutrophils, recruited to the site of infection, eliminate microorganisms by phagocytosis, degranulation, and generation of reactive oxygen species (ROS)1. In the presence of bacteria or endotoxins, neutrophils release neutrophil extracellular traps (NETs), composed of extracellular chromatin decorated with histones and granular proteins like elastase and myeloperoxidase (MPO)2. Although NETs have indispensable antimicrobial properties, increasing experimental and clinical evidence suggests that overzealous NET formation during sepsis may lead to multiple organ dysfunction and death3,4,5,6.
Because NETs may play a similar pathophysiological role in dogs, therapeutic interventions that either prevent or diminish NET formation may serve as novel treatment strategies in septic animals. For this reason, there is a need for a reliable technique to assess and quantify NETosis and NET components in dogs. NET components including cell-free DNA (cfDNA) and nucleosomes have previously been evaluated in canine neutrophils and plasma from clinical dogs7,8,9. Using fluorescence assays, Goggs and Letendre found that septic dogs have higher levels of cfDNA than healthy dogs8. Although these techniques are highly objective and quantitative, measurement of cfDNA and nucleosomes as markers of NETosis is non-specific since they can be derived from necrotic cells other than NETosing neutrophils. Here we describe a technique that utilizes fluorescence microscopy to examine the behaviours of live NETosing neutrophils. We also detail a modified protocol using double-immunolabeling to subjectively quantify NETs and their components such as MPO and citH3 in canine neutrophils10.

<table><tbody><tr><td>Dextran from &lt;em&gt;Leuconostoc&lt;/em&gt; spp.</td><td>Sigma</td><td>31392</td><td>Molecular weight 450,000 &amp;ndash; 650,000</td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Life Sciences</td><td>17144002</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline</td><td>ThermoFisher Scientific</td><td>A1285801</td><td>With divalent cations</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline</td><td>ThermoFisher Scientific</td><td>14190136</td><td>Without divalent cations</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; O55:B5</td><td>InvivoGen</td><td></td><td></td></tr><tr><td>SYTOX Orange Fluorescent Nucleic Acid Stain</td><td>ThermoFisher Scientific</td><td>S11368</td><td>5 mM in DMSO; stains in cells with permeable membranes</td></tr><tr><td>SYTO Green 16 Fluorescent Nucleic Acid Stain</td><td>ThermoFisher Scientific</td><td>S7578</td><td>1 mM in DMSO; stains in cells with intact membranes</td></tr><tr><td>Surface-Amps NP-40</td><td>Pierce</td><td>28324</td><td></td></tr><tr><td>Poly-D-Lysine coated coverslips</td><td>neuVitro</td><td>H-12-pdl</td><td>12 mm diameter</td></tr><tr><td>Anti-citrullinated histone H3 antibody</td><td>Abcam</td><td>Ab5103</td><td></td></tr><tr><td>Unconjugated goat anti-rabbit Fab fragments</td><td>Jackson ImmunoResearch</td><td>111-007-003</td><td>Specificity: IgG (H+L)</td></tr><tr><td>Anti- Myeloperoxidase antibody</td><td>Dako</td><td>A0398</td><td></td></tr><tr><td>4,6-Diamidino-2-phenylin (DAPI)</td><td>Life Technologies Corporation</td><td>D1306</td><td></td></tr></tbody></table>

in vitro canine neutrophil extracellular trap formation: dynamic and quantitative analysis by fluorescence microscopy

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

Using this protocol of live cell imaging, investigators can observe the nuclear morphology, plasma membrane integrity and presence of cfDNA in living neutrophils. A cell impermeant nuclear dye stains nuclei acids red in cells with damaged cell membranes. Another cell-permeant dye, labels intracellular nucleic acids in live cells with intact plasma membranes. All intact neutrophils, regardless of their treatments, should appear green and exhibit the characteristic lobulated nuclei at 0 to ...

Watch this Scientific Journal Video about In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy at JoVE.com

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

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

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

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

in-vitro-canine-neutrophil-extracellular-trap-formation-dynamic

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Immunology and Infection

8.7K Views.  University of California, Davis. We beschrijven de methoden om te isoleren canine neutrofielen van volbloed en visualiseren van netto vorming in levende neutrofiele granulocyten met behulp van fluorescentie microscopie. Ook beschreven zijn protocollen te kwantificeren netto vorming en citrullinated Histon H3 (citH3)-expressie met immunofluorescentie microscopie.

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

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 ...

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

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

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

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

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 ...

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

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

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.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Medicine

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 ...

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.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

Real-Time Microscopy for Studying NETosis Kinetics and Screening Inhibitors

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

This defined protocol describes a real-time high-throughput microscopy approach to visualize and quantify human neutrophil extracellular trap (NET) release in vitro. The reproducible method allows investigation of the characteristics and kinetics of NET release upon stimulation with distinct NETosis inducers and enables assessment of the pharmacology of NETosis antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in ...

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

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