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. J., Radic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+Double-edged+swords+of+innate+immunity.">Neutrophil extracellular traps: Double-edged swords of innate immunity.</a> The Journal of Immunology. 189 (6), 2689-2695 (2012).</li><li>Czaikoski, P. G., 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=Neutrophil+Extracellular+Traps+Induce+Organ+Damage+during+Experimental+and+Clinical+Sepsis.">Neutrophil Extracellular Traps Induce Organ Damage during Experimental and Clinical Sepsis.</a> PLoS One. 11 (2), e0148142 (2016).</li><li>Dwivedi, D. J., 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=Prognostic+utility+and+characterization+of+cell-free+DNA+in+patients+with+severe+sepsis.">Prognostic utility and characterization of cell-free DNA in patients with severe sepsis.</a> Critical Care. 16 (4), R151 (2012).</li><li>Mai, S. H., 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=Delayed+but+not+Early+Treatment+with+DNase+Reduces+Organ+Damage+and+Improves+Outcome+in+a+Murine+Model+of+Sepsis.">Delayed but not Early Treatment with DNase Reduces Organ Damage and Improves Outcome in a Murine Model of Sepsis.</a> Shock. 44 (2), 166-172 (2015).</li><li>Jeffery, U., 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=Dogs+cast+NETs+too:+Canine+neutrophil+extracellular+traps+in+health+and+immune-mediated+hemolytic+anemia.">Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia.</a> Veterinary Immunology and Immunopathology. 168 (3-4), 262-268 (2015).</li><li>Letendre, J. A., Goggs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+plasma+cell-free+DNA+concentrations+in+dogs+with+sepsis,+trauma,+and+neoplasia.">Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia.</a> Journal of Veterinary Emergency and Critical Care. 27 (3), 307-314 (2017).</li><li>Jeffery, U., Gray, R. D., LeVine, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Fluorescence+Assay+for+Quantification+of+Canine+Neutrophil+Extracellular+Trap+Release.">A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release.</a> J Vis Exp. (117), (2016).</li><li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+how+to+generate+and+visualize+them.">Neutrophil extracellular traps: how to generate and visualize them.</a> Journal of Visualized Experiments. (36), (2010).</li><li>Oh, H., Siano, B., Diamond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+isolation+protocol.">Neutrophil isolation protocol.</a> Journal of Visualized Experiments. 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L., Ng, G., Tablin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+neutrophil+extracellular+trap+formation+in+canine+neutrophils+is+dependent+on+histone+H3+citrullination+by+peptidylarginine+deiminase.">Lipopolysaccharide-induced neutrophil extracellular trap formation in canine neutrophils is dependent on histone H3 citrullination by peptidylarginine deiminase.</a> Veterinary Immunology and Immunopathology. 193, 29-37 (2017).</li><li>Masuda, S., 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=NETosis+markers:+Quest+for+specific,+objective,+and+quantitative+markers.">NETosis markers: Quest for specific, objective, and quantitative markers.</a> Clinica Chimica Acta. 459, 89-93 (2016).</li><li>Li, P., 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=PAD4+is+essential+for+antibacterial+innate+immunity+mediated+by+neutrophil+extracellular+traps.">PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps.</a> Journal of Experimental Medicine. 207 (9), 1853-1862 (2010).</li><li>Leshner, M., 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=PAD4+mediated+histone+hypercitrullination+induces+heterochromatin+decondensation+and+chromatin+unfolding+to+form+neutrophil+extracellular+trap-like+structures.">PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures.</a> Frontiers in Immunology. 3, 307 (2012).</li><li>Luo, Y., 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=Inhibitors+and+inactivators+of+protein+arginine+deiminase+4:+functional+and+structural+characterization.">Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization.</a> Biochemistry. 45 (39), 11727-11736 (2006).</li></ol>

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 Leuconostoc spp.</td>
			<td>Sigma</td>
			<td>31392</td>
			<td>Molecular weight 450,000 &#8211; 650,000</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Life Sciences</td>
			<td>17144002</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>A1285801</td>
			<td>With divalent cations</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Saline</td>
			<td>ThermoFisher Scientific</td>
			<td>14190136</td>
			<td>Without divalent cations</td>
		</tr>
		<tr>
			<td>E. coli 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

8.6K Views.  University of California, Davis. 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.

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 ...

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

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

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

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

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

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

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

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

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

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

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

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

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

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

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. ...