This work was supported by a basic science grant (#285480) from CONACyT and by the Department of Clinical Immunology Research of the Biochemical Sciences Faculty, Universidad Aut&#243;noma &#39;Benito Ju&#225;rez&#39; de Oaxaca. A.A.A, S.A.S.L, and W.J.R.R. have doctoral fellowships of CONACyT numbers #799779, #660793, and #827788, respectively.

The blood samples were obtained as donations from clinically healthy participants after informed consent. All experiments were performed with the permission of the Human Research Ethics Committee of the Faculty of Biochemical Sciences, Universidad Aut&#243;noma &#39;Benito Ju&#225;rez&#39; of Oaxaca.
NOTE: The inclusion criteria in the study were indistinct sex and age, and clinically healthy according to participant responses to a questionnaire prior to taking a blood sample. A hematological analysis was performed to determine the cell count and rule out infections or anemia, as well as the C-reactive protein test to rule out inflammation in the donor.
1. Peripheral blood collection and obtaining the erythrocyte and leukocyte package
<ol>
	<li>Collect 10 mL of peripheral blood by venipuncture in tubes with 1.8 mg/mL of K2&#183;EDTA as anticoagulant (see Table of Materials) from clinically healthy individuals after obtaining informed consent. Then, perform standard blood biometry and C-reactive protein test to rule out infection or inflammation, ensuring the quality of the sample.</li>
	<li>Centrifuge the peripheral blood sample at 82 x g for 15 min to remove the platelet-rich plasma, followed by a second centrifugation at 630 x g for 5 min. Discard the remaining plasma to obtain the erythrocyte and leukocyte package.</li>
	<li>Dilute it in 1:1 ratio (v/v) with 1x Dulbecco&#39;s phosphate-buffered saline (DPBS).</li>
</ol>
2. Polymorphonuclear neutrophil (PMN) purification using a double-density gradient
NOTE: Perform neutrophil purification immediately after the blood is collected, because they have a limited in vitro lifetime of about 8 h.
<ol>
	<li>Deposit the following in a sterile 10 mL glass tube (see Table of Materials) in order: 1 mL of 1.098 g/mL density solution, 1 mL of 1.079 g/mL density solution (see Table of Materials), and then 4 mL of the diluted erythrocyte and leukocyte package. Pour over the walls without breaking the surface tension between the layers to prevent them from mixing.</li>
	<li>Centrifuge at 320 x g for 20 min at 4 &#176;C, avoiding acceleration/deceleration so that the high forces of the centrifuge do not disturb the gradient.</li>
	<li>Aspirate the phase that corresponds to granulocytes (Figure 1A) by pipetting, and transfer to another sterile 10 mL glass tube. Wash with 4 mL of 1x DPBS at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Discard the supernatant and treat the cells with osmotic shock to remove the remaining erythrocytes. Add 4 mL of 0.2% saline solution for 2 min at 4 &#176;C, and centrifuge at 300 x g for 10 min at 4 &#176;C. Discard the supernatant. Then add 4 mL of the isotonic solution (0.65% saline) for 5 min at 4 &#176;C to restore membrane integrity, and centrifuge at 300 x g for 10 min at 4 &#176;C. 
	NOTE: The 0.2% saline solution is a hypotonic medium, with a lower solute concentration relative to that of the RBC intracellular medium. Contact with the hypotonicmedium allows water to diffuse into the RBC, leading to their swelling and hemolysis. This removal of RBC from the supernatant was confirmed by microscopic observation.</li>
	<li>Remove the supernatant. Resuspend the cells in 4 mL of 1x DPBS to remove cellular debris, and then centrifuge at 300 x g for 10 min at 4 &#176;C. Finally, resuspend the cell pellet in 2 mL of cold Hank&#39;s balanced salt solution (HBSS) buffer.</li>
</ol>
3. Neutrophil morphology and viability (Figure 1B)
<ol>
	<li>Trypan blue exclusion test
	<ol>
		<li>Dilute 5 &#956;L of the cell suspension in 20 &#956;L of 0.4% trypan blue (1:5 ratio). Count the cells in a Neubauer chamber and determine cell viability using an exclusion test. Consider the cells that maintain the integrity of their membrane without permeabilizing the dye as viable.</li>
		<li>Mount 5 &#956;L of the cell suspension on a slide; dry and stain with Wright&#39;s stain for 15 s. Immediately fix the sample with phosphate buffer pH 6.4 for 30 s. Wash with sufficient distilled water and observe the morphology under an optical microscope (100x).</li>
	</ol>
	</li>
	<li>7AAD-staining and flow cytometry analysis
	<ol>
		<li>Add 1 x 105 cells to flow cytometry tubes, and stain with 1 &#956;L of 7AAD in 100 &#956;L of FACS buffer (1x DPBS, 0.1% sodium azide, and 10% autologous decomplemented plasma) for 15 min at 4 &#176;C in the dark.</li>
		<li>Wash with 500 &#956;L of FACS buffer at 300 x g for 10 min. Fix the cells with 500 &#956;L of 2% paraformaldehyde, and store at 4 &#176;C until their analysis in the flow cytometer.</li>
		<li>For a dead cell control, fix 1 x 105 cells with 200 &#956;L of 4% paraformaldehyde for 30 min, and wash with 500 &#956;L of 1x PBS at 300 x g for 10 min at 4 &#176;C. Draw off the supernatant and discard. Then add 200 &#956;L of 0.1% Triton X-100 for 1 h at 4 &#176;C. Wash with 500 &#956;L of 1x PBS and stain with 7AAD as in step 3.2.1.</li>
		<li>Using a flow cytometer (see Table of Materials), perform FSC versus SSC analysis to analyze cell purity and SSC versus 7AAD staining to analyze the cell viability. Read 3 x 104 events in 100 &#956;L of uptake volume at medium flow (1,000 cells/s) in the polymorphonuclear settings (FSC, 400-490 and SSC, 300-320).</li>
		<li>Analyze the captured data in the flow cytometer software (see Table of Materials), and determine the percentage of purity and positive cells for 7AAD in the polymorphonuclear population, presented through dot plots and histograms.</li>
	</ol>
	</li>
</ol>
4. CFSE staining of microorganisms
<ol>
	<li>Add 1 x 108 bacteria or 1 x 106 fungal pseudohyphae in 1.5 mL microtubes, and stain with 200 &#181;L of 5 &#181;M carboxyfluorescein succinimidyl ester (CFSE) dissolved in 1x PBS. Mix for a few seconds, and incubate at 37 &#176;C for 10 min in the dark.</li>
	<li>Stop the reaction by adding 500 &#181;L of decomplemented plasma, and centrifuge at 620 x g for 10 min for pseudohyphae or at 1,800 x g for 10 min for bacteria.</li>
	<li>Discard the supernatants and wash the pellets with 1 mL of 1x PBS with centrifugation as in step 4.2. Finally, resuspend the microorganisms in 250 &#181;L of 1x PBS.</li>
	<li>Prepare 50 &#181;L aliquots in microtubes of 1.5 mL with 2 x 107 bacteria (MOI: 100) or 2 x 105 pseudohyphae (MOI:1) for NET induction.</li>
</ol>
5. NET induction
<ol>
	<li>Place 10 mm x 10 mm sterile glass coverslips in a 24-well plate and cover with 10 &#181;L of 0.001% poly-L-lysine for 1 h at room temperature. Wash twice with 100 &#956;L of 1x PBS, air dry, and irradiate with UV light for 15 min.</li>
	<li>Replace the HBSS solution of the neutrophil suspension in step 2.5 with RPMI 1640 medium supplemented with 10%&#160;autologous plasma. To the 24-well plate (step 5.1), add 350 &#956;L of this cell suspension, for a final concentration of 2 x 105 neutrophils/well.</li>
	<li>Allow the cells to adhere to the bottom of the wells by incubating for 20 min at 37 &#176;C with 5% CO2.</li>
	<li>Add the stimuli to induce NET formation in 50 &#181;L: microbial stimuli-gram-positive bacterium S. aureus (ATCC 25923), gram-negative bacterium P. aeruginosa (ATCC 10145) at MOI 100, and pseudohyphae of C. albicans (ATCC 10231) at MOI:1; biochemical stimuli-PMA (200 nM) and HOCl (4.5 mM), and control with stimulus absent (50 &#181;L of HBSS).</li>
	<li>Obtain a final volume of 400 &#181;L per well. Mix on a plate shaker at 140 rpm for 30 s, and incubate for 4 h at 37 &#176;C and 5% CO2.</li>
</ol>
6. Visualization of NETs by fluorescence microscopy
<ol>
	<li>DNA and LL37 immunostaining
	<ol>
		<li>After NET induction, remove the supernatants from the wells by pipetting carefully, and fix the cells with 300 &#181;L of 4% paraformaldehyde for 30 min.</li>
		<li>Wash the cells with 200 &#956;L of 1x PBS without centrifuging, and add 200 &#181;L of blocking buffer (10% decomplemented plasma in 1x PBS) for 30 min.</li>
		<li>For LL-37 stain, permeabilize the cells with 200 &#181;L of 0.2% Triton X-100 in 1x PBS for 10 min to allow the antibody to enter the cells. Wash 2x carefully with 1x PBS to remove the excess detergent.</li>
		<li>Mount the coverslips on glass slides (four coverslips on each slide). DNA stain the cells with 2 &#181;L of DAPI (see Table of Materials), seal the coverslips, and store at -20 &#176;C until their analysis by confocal fluorescence microscopy.</li>
	</ol>
	</li>
	<li>Acquisition and analysis of fluorescent images
	<ol>
		<li>Take NET images to quantify their components, and use the corresponding filters in the confocal fluorescence microscope (see Table of Materials) to acquire the images with the computer&#39;s software. 
		NOTE: Consider that the DNA is stained with DAPI (blue color), showing excitation at 360 nm and emission at 460 nm. The microorganisms are stained with CFSE (green color), which has an excitation of 492 nm and an emission of 521 nm. The LL37 peptide is labeled with anti-LL37 Alexa Fluor 594 antibody (red color), which has an excitation of 594 nm and an emission of 614 nm.</li>
		<li>Calibrate the microscope. Place the slide and focus using differential interference contrast (DIC) with normal light on. Choose Live to project the image on the monitor.</li>
		<li>Turn off the light and select the fluorochrome corresponding channel. For example, select filter 365 nm/blue for DAPI, 43 HE DsRed for Alexa 594, or 38 HE GFP for CFSE.</li>
		<li>Adjust the settings with the isotype control antibody for LL37 and unstained cells for DAPI and CFSE. Set the same exposure time, voltage, contrast, and lens settings to capture all the images under the same conditions. 
		NOTE: In this study, the exposure time, voltage, and contrast were set at 1.0 ms, 4.0 V, and 0.0, respectively, with a 40x objective. These values can be adjusted to facilitate the best image capture for the samples.</li>
		<li>Select Snap to capture the image. Save five images (four extremes and the center) per well, and of the colocalization (merge) of DNA/LL37/CFSE.</li>
		<li>Define the three classes of pixels as background with the independent images of each color and analyze the Mean Gray Signal value per area with the Image J software.</li>
	</ol>
	</li>
</ol>
7. Enzymatic activity quantification
<ol>
	<li>In a 96-well plate, add 90 &#956;L of cell suspension in HBSS containing 1 x 105 neutrophils for NET induction, and incubate for 20 min at 37 &#176;C and 5% CO2.</li>
	<li>Immediately, add 10 &#956;L of the corresponding stimuli (concentration as in step 5.4) and incubate for 4 h at 37 &#176;C with 5% CO2.</li>
	<li>Discard the supernatants and wash the cells with 100 &#956;L of HBSS. Treat with 1 U/mL of DNase for 10 min at 37 &#176;C to favor the release of DNA-protein structures, and centrifuge at 1,800 x g for 10 min.</li>
	<li>Recover the supernatants and evaluate the enzyme activity in the supernatant using colorimetric reactions as previously described by White et al.17.</li>
	<li>Determine the maximum enzyme activity of NE, CG, and MPO in neutrophils under the same experimental conditions without adding any stimuli for NET induction. Then, freeze the cell sample at -70 &#176;C and thaw at 37 &#176;C in a water bath, generating a temperature shock to favor the release of intracell proteins by cell lysis. Centrifuge at 1,800 x g for 10 min and recover the supernatants.</li>
	<li>Add 50 &#956;L of the supernatant to each well in 96-well plates, and then add 50 &#956;L of each substrate as indicated in step 7.7.</li>
	<li>Add 0.5 M of N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitro aniline as the substrate for NE, and 1 mM of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide for CG. Incubate for 3 h at room temperature. For MPO, add 1.6 mM of 3,3&#39;, 5,5&#39;-tetramethylbenzidine (TMB) and incubate for 30 min at room temperature.</li>
	<li>Post-incubation, add 50 &#956;L of the stop solution (0.5 M H2SO4) for MPO and measure the absorbance at 405 nm for NE and CG and 450 nm for MPO, using a spectrophotometer.</li>
	<li>Compare the values obtained with the corresponding calibration curves and show the results of each condition relative to the maximum enzyme activity (100%).</li>
</ol>
8. Statistical analysis
<ol>
	<li>Analyze the measurement data in triplicate for each independent experiment (n = 10) and perform an ANOVA for statistical analysis by comparing groups with a 95% confidence level.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64522/64522fig01.jpg" /> 
Figure 1: PMN purification and NET induction protocol. (A) Plasma was removed from the peripheral blood to obtain the erythrocyte and leukocyte package and diluted 1:1 (v/v) with 1x DPBS. Then, 4 mL of the dilution was added along the wall to the double-density gradient tube, and centrifuged at 320 x g for 20 min at 4 &#176;C, obtaining the separation of different cell layers and recovering the one corresponding to PMN. (B) The purified cells were counted, and their morphology was analyzed by Wright&#39;s staining. Viability was determined by trypan blue exclusion and 7AAD staining using flow cytometry. Once optimal neutrophil purity and viability were verified, NET formation was induced by adding microbes (S. aureus, P. aeruginosa, and C. albicans) or chemicals (PMA, HOCl) in 24-well plates for analysis by fluorescence microscopy with DAPI-DNA, anti-LL37 Alexa Fluor 594, and microorganism-CFSE staining. For enzyme quantification, NETs were induced in 96-well plates for 3 h and treated with DNase, followed by the addition of substrates for each enzyme: NE, CG, and MPO; color changes were quantified by spectrophotometry. DPBS = Dulbecco&#39;s phosphate-buffered saline; PBMC = Peripheral blood mononuclear cells; PMN = Polymorphonuclear neutrophils; NE = Neutrophil elastase; CG = Cathepsin G; MPO = Myeloperoxidase; PMA = Phorbol myristate acetate; HOCl = Hypochlorous acid. <a href="https://www.jove.com/files/ftp_upload/64522/64522fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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C., 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=Characterization,+quantification,+and+visualization+of+neutrophil+extracellular+traps.">Characterization, quantification, and visualization of neutrophil extracellular traps.</a> Methods in Molecular Biology. 1537, 481-497 (2017).</li><li>Boeltz, 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=To+NET+or+not+to+NET:+current+opinions+and+state+of+the+science+regarding+the+formation+of+neutrophil+extracellular+traps.">To NET or not to NET: current opinions and state of the science regarding the formation of neutrophil extracellular traps.</a> Cell Death and Differentiation. 26 (3), 395-408 (2019).</li><li>Yousefi, S., Mihalache, C., Kozlowski, E., Schmid, I., Simon, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viable+neutrophils+release+mitochondrial+DNA+to+form+neutrophil+extracellular+traps.">Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps.</a> Cell Death and Differentiation. 16 (11), 1438-1444 (2009).</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=Neutrophil+extracellular+traps:+is+immunity+the+second+function+of+chromatin.">Neutrophil extracellular traps: is immunity the second function of chromatin.</a> The Journal of Cell Biology. 198 (5), 773-783 (2012).</li></ol>

The authors declare that they have no conflicts of interest.

A highly pure population of viable neutrophils must be obtained to induce the release of NETs since these cells have a limited ex vivo lifetime of 8 h on average, a period within which all the experiments must be performed. To this end, the ideal methodology is the double-density gradient to optimize the purification time by isolating nonactivated cells more responsive to exogenous stimulation, in contrast to Ficoll-Histopaque gradient or Dextran sedimentation techniques17. Another advant...

Neutrophils are the most abundant leukocytes in the bloodstream, playing an essential role during the clearance of pathogenic agents by several mechanisms, including the release of large chromatin structures composed of DNA and several nuclear, cytoplasmic, and granular antibacterial proteins1,2. The direct antecedent describing this antimicrobial role of neutrophils was made by Takei et al.3 in 1996. These authors reported a new form of death different from apoptosis and necroptosis in neutrophils, showed morphological changes exhibiting nuclear rupture, followed by spilling out of the nucleoplasm into the cytoplasm, and an increase in membrane permeability from 3 h of incubation with phorbol myristate acetate (PMA)2,3. However, it was not until 2004 that the term &#34;neutrophil extracellular traps (NETs)&#34; was used4.
NET formation has been observed in various conditions, such as bacterial, fungal5, viral6, and parasitic infections, for neutralizing, killing, and preventing microbial dissemination7. Other studies show that it can also occur in non-pathogenic conditions by sterile stimuli, such as cytokines, monosodium uric acid or cholesterol crystals, autoantibodies, immune complexes, and activated platelets7. Lipopolysaccharide (LPS), interleukin-8 (IL-8), and PMA were among the first in vitro stimuli described as NET inducers, and the in vivo NET involvement in pathogenic processes was demonstrated in two models of acute inflammation: experimental dysentery and spontaneous human appendicitis4. DNA is an essential NET component. Its appropriate structure and composition are necessary for the sequestration and killing of microorganisms by delivering a high local concentration of antimicrobial molecules toward the caught microbes, as demonstrated by a brief deoxyribonuclease (DNase) treatment that disintegrates NETs and their microbicidal properties4. Besides DNA, NETs comprise attached proteins such as histones, neutrophil elastase (NE), cathepsin G (CG), proteinase 3, lactoferrin, gelatinase, myeloperoxidase (MPO), and antimicrobial peptides (AMPs) such as the cationic pro-inflammatory peptide cathelicidin LL-37 among others8,9. Such aggregates may form larger threads with diameters up to 50 nm. These factors can disrupt the microbial virulence factors or the integrity of the pathogen cell membrane; additionally, the AMPs can stabilize the NET-derived DNA against degradation by bacterial nucleases10.
The specific mechanisms regulating NET formation have not yet been completely clarified. The best-characterized pathway leading to NET release is through ERK signaling, which leads to NADPH oxidase activation and reactive oxygen species (ROS) production, as well as increased intracellular calcium that triggers activation of the MPO pathway. This in turn transforms hydrogen peroxide into hypochlorous acid, activating NE by oxidation11,12. NE is responsible for degrading the actin filaments of the cytoskeleton to block phagocytosis and translocating them to the nucleus for processing by proteolytic cleavage and deamination by PAD4 that drive the desensitization of chromatin fibers, which associate with granule and cytoplasmic proteins, and are then released extracellularly7. These proteases include those released from the azurosome complex of the azurophil granules and other proteases such as cathepsin G13.
Depending on the morphological changes in neutrophils, NETs are classified into two types: suicidal or lytic NET formation leading to cell death4, and vital or non-lytic NET formation produced by viable cells mediated by a vesicular release of nuclear or mitochondrial DNA, with a remnant of an anucleated cytoplast with phagocytic capability14,15. Generally, NETs composed of mitochondrial DNA present an elongated fiber14 morphology, while those structured of nuclear DNA have a cloud-like appearance3. However, it is not known how the neutrophil chooses its DNA origin. Contrary to previous studies that described the canonical pathways of NETs as requiring several hours, the vital pathway is rapidly activated in just 5-60 min15.
Despite these advances, the NET composition varies depending on the stimulus; for example, different mucoid and non-mucoid strains of P. aeruginosa induce the formation of NETs containing 33 common proteins and up to 50 variable proteins7. Thus, it is necessary to homogenize techniques that allow the generation of objective conclusions in research groups. This paper describes a protocol with various techniques that allow comparison and evaluation of the composition, structure, and morphology of NETs induced with different microorganisms: Staphylococcus aureus (gram-positive bacterium), Pseudomonas aeruginosa (gram-negative bacterium), and Candida albicans (fungus), as well as chemical stimuli (PMA, HOCl) in human neutrophils from healthy individuals. The representative results demonstrate the heterogeneity of NETs depending on their inducing stimulus under comparable in vitro conditions, characterized by DNA-DAPI staining, immunostaining for LL37, and quantification of enzymatic activity (NE, CG, and MPO).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 Well plate for cell culture</td>
			<td>Corning&#160;</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>7-aminoactinomycin D (7-AAD)</td>
			<td>BD Pharmingen</td>
			<td>51-668981E</td>
			<td></td>
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			<td>96 Well plate for cell culture</td>
			<td>Costar</td>
			<td>3596</td>
			<td>Flat bottom</td>
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			<td>Agitator</td>
			<td>CRM Globe</td>
			<td>CRM-OS1&#160;</td>
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			<td>Antibody LL37&#160;&#160;</td>
			<td>Santa Cruz Biotechnology&#160;</td>
			<td>sc-166770</td>
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		</tr>
		<tr>
			<td>Blood collection tubes</td>
			<td>BD VACUTAINER</td>
			<td>368171</td>
			<td>K2 EDTA 7.2 mg</td>
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		<tr>
			<td>Carboxyfluorescein succinimidyl ester (CFSE)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>21878</td>
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		<tr>
			<td>Centrifuge</td>
			<td>Hettich</td>
			<td>1406-01</td>
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			<td>Coverslip</td>
			<td>Madesa</td>
			<td>M03-CUB-22X22</td>
			<td>22 mm x 22 mm</td>
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		<tr>
			<td>Dulbecco&#180;s phosphate-buffered saline (DPBS)</td>
			<td>Caisson</td>
			<td>1201022</td>
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			<td>Falcon tubes 50 mL</td>
			<td>CORNING&#160;</td>
			<td>430829</td>
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		<tr>
			<td>Flow Cytometry Tubes</td>
			<td>Miltenyi Biotec&#160;</td>
			<td></td>
			<td>5 mL - Without caps</td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Analyze flow cytometry data</td>
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			<td>Fluorescence microscope</td>
			<td>DM 2000&#160;</td>
			<td>LEICA&#160;</td>
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			<td>Fluoroshield with DAPI&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F6057&#160;</td>
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			<td>Incubator&#160;</td>
			<td>NUAIRE</td>
			<td>UN-4750</td>
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			<td>MACSQuant Analyzer</td>
			<td>Miltenyi Biotec&#160;</td>
			<td></td>
			<td>Flow cytometer</td>
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			<td>Microplate reader photometer&#160;</td>
			<td>Clarkson Laboratory - CL&#160;</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Microtubes 1.5 mL</td>
			<td>Zhejiang Runlab Tech</td>
			<td>35200N</td>
			<td>wire snap&#160;</td>
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		<tr>
			<td>Minitab Software</td>
			<td>Minitab</td>
			<td></td>
			<td>Statistical analysis</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>BD VACUTAINER</td>
			<td>301746</td>
			<td>Diameter 1.34 mm</td>
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		<tr>
			<td>Optical microscope</td>
			<td>VELAB</td>
			<td>VE-B50</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td>Solution for density gradient</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10x)</td>
			<td>Caisson</td>
			<td>PBL07-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex culture tubes</td>
			<td>CORNING&#160;</td>
			<td>CLS982025</td>
			<td>N&#176;9820</td>
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		<tr>
			<td>RPMI 1640 1x</td>
			<td>Corning&#160;</td>
			<td>10-104-CV</td>
			<td>contains Glutagro</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>Madesa</td>
			<td>PDI257550</td>
			<td>22 mm x 75 mm</td>
		</tr>
		<tr>
			<td>Trypan Blue solution 0.4%</td>
			<td>SIGMA</td>
			<td>T8154-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Purity and viability of neutrophils 
The dynamic cellular phases are visualized in the tube from the double-density gradient purification. Within these layers, the layer corresponding to granulocytes is above the 1.079 g/mL density layer, distinguished from the phases of peripheral blood mononucleocytes (PBMCs) and erythrocytes (Figure 1A). The morphology of the purified cells was verified with Wright&#39;s staining by observing cells with segmented nuclei connected wit...

Watch this Scientific Journal Video about Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli at JoVE.com

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

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 ...

morphological-compositional-analysis-neutrophil-extracellular-traps

Research

JoVE Journal

Immunology and Infection

2.2K Views.  Universidad Autónoma “Benito Juárez” de Oaxaca. 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.

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

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 ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

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

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

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

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

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

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

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.

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

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 ...

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 ...