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 ...

<ol>
	<li>De Buhr, N., Maren, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+neutrophil+extracellular+traps+become+visible.">How neutrophil extracellular traps become visible.</a> Journal of Immunology Research. 2016, 4604713 (2016).</li><li>Karlsson, A., Nixon, J. B., McPhail, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phorbol+myristate+acetate+induces+neutrophil+NADPH-oxidase+activity+by+two+separate+signal+transduction+pathways:+dependent+or+independent+of+phosphatidylinositol+3-kinase.">Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase.</a> Journal of Leukocyte Biology. 67 (3), 396-404 (2000).</li><li>Takei, H., Araki, A., Watanabe, H., Ichinose, A., Sendo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+killing+of+human+neutrophils+by+the+potent+activator+phorbol+12-myristate+13-acetate+(PMA)+accompanied+by+changes+different+from+typical+apoptosis+or+necrosis.">Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis or necrosis.</a> Journal of Leukocyte Biology. 59 (2), 229-240 (1996).</li><li>Brinkmann, V., 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+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Kenny, E. F., 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=Diverse+stimuli+engage+different+neutrophil+extracellular+trap+pathways.">Diverse stimuli engage different neutrophil extracellular trap pathways.</a> eLife. 6, 24437 (2017).</li><li>Schultz, B. M., Acevedo, O. A., Kalergis, A. M., Bueno, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+extracellular+trap+release+during+bacterial+and+viral+infection.">Role of extracellular trap release during bacterial and viral infection.</a> Frontiers in Microbiology. 13, 798853 (2022).</li><li>Papayannopoulos, V. <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+in+immunity+and+disease.">Neutrophil extracellular traps in immunity and disease.</a> Nature Reviews Immunology. 18 (2), 134-147 (2018).</li><li>Delgado-Rizo, V., 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+and+its+implications+in+inflammation:+An+overview.">Neutrophil extracellular traps and its implications in inflammation: An overview.</a> Frontiers in Immunology. 8, 81 (2017).</li><li>Petretto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+(NET)+induced+by+different+stimuli:+A+comparative+proteomic+analysis.">Neutrophil extracellular traps (NET) induced by different stimuli: A comparative proteomic analysis.</a> PLoS One. 14 (7), 0218946 (2019).</li><li>Neumann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+role+of+the+antimicrobial+peptide+LL-37+in+the+protection+of+neutrophil+extracellular+traps+against+degradation+by+bacterial+nucleases.">Novel role of the antimicrobial peptide LL-37 in the protection of neutrophil extracellular traps against degradation by bacterial nucleases.</a> Journal of Innate Immunity. 6 (6), 860-868 (2014).</li><li>Hakkim, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Raf-MEK-ERK+pathway+is+required+for+neutrophil+extracellular+trap+formation.">Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation.</a> Nature Chemical Biology. 7 (2), 75-77 (2011).</li><li>Sabbatini, M., Magnelli, V., Ren&#242;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NETosis+in+wound+healing:+When+enough+Is+enough.">NETosis in wound healing: When enough Is enough.</a> Cells. 10 (3), 494 (2021).</li><li>Metzler, K. D., Goosmann, C., Lubojemska, A., Zychlinsky, A., Papayannopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myeloperoxidase-containing+complex+regulates+neutrophil+elastase+release+and+actin+dynamics+during+NETosis.">A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis.</a> Cell Reports. 8 (3), 883-896 (2014).</li><li>Clark, S. R., 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=Platelet+TLR4+activates+neutrophil+extracellular+traps+to+ensnare+bacteria+in+septic+blood.">Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood.</a> Nature Medicine. 13 (4), 463-469 (2007).</li><li>Pilsczek, F. 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=A+novel+mechanism+of+rapid+nuclear+neutrophil+extracellular+trap+formation+in+response+to+Staphylococcus+aureus.">A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus.</a> Journal of Immunology. 185 (12), 7413-7425 (2010).</li><li>Sosa, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+differences+of+neutrophil+extracellular+traps+induced+by+biochemical+and+microbiologic+stimuli+under+healthy+and+autoimmune+milieus.">Structural differences of neutrophil extracellular traps induced by biochemical and microbiologic stimuli under healthy and autoimmune milieus.</a> Immunologic Research. 69 (3), 264-274 (2021).</li><li>White, P. 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).

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			<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>
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			<td>96 Well plate for cell culture</td>
			<td>Costar</td>
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			<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>
			<td></td>
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			<td>Blood collection tubes</td>
			<td>BD VACUTAINER</td>
			<td>368171</td>
			<td>K2 EDTA 7.2 mg</td>
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			<td>Carboxyfluorescein succinimidyl ester (CFSE)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>21878</td>
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			<td>Centrifuge</td>
			<td>Hettich</td>
			<td>1406-01</td>
			<td></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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			<td>Dulbecco&#180;s phosphate-buffered saline (DPBS)</td>
			<td>Caisson</td>
			<td>1201022</td>
			<td></td>
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		<tr>
			<td>Falcon tubes 50 mL</td>
			<td>CORNING&#160;</td>
			<td>430829</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>DM 2000&#160;</td>
			<td>LEICA&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F6057&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>NUAIRE</td>
			<td>UN-4750</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQuant Analyzer</td>
			<td>Miltenyi Biotec&#160;</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>Microplate reader photometer&#160;</td>
			<td>Clarkson Laboratory - CL&#160;</td>
			<td></td>
			<td></td>
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			<td>Microtubes 1.5 mL</td>
			<td>Zhejiang Runlab Tech</td>
			<td>35200N</td>
			<td>wire snap&#160;</td>
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			<td>Minitab Software</td>
			<td>Minitab</td>
			<td></td>
			<td>Statistical analysis</td>
		</tr>
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			<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>
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			<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>
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			<td>Pyrex culture tubes</td>
			<td>CORNING&#160;</td>
			<td>CLS982025</td>
			<td>N&#176;9820</td>
		</tr>
		<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>
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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 ...

This protocol demonstrates the heterogeneity in composition, structure, and morphology of neutrophil extracellular traps, depending on their inducing stimulus under comparable in vitro conditions in human neutrophils from healthy individuals. High neutrophil purity and viability are obtained. That allows to compare the concentration of DNA, LL-37, and enzyme activity of myeloperoxidase, elastase, and cathepsin G, released by neutrophil extracellular traps.This protocol allowed to analyze the effect of soluble factors or cellular contact or possible therapies or mechanisms for closing on the production of NETs in autoimmune disease. These methods can aid in the investigation of autoimmune disease. Due to the uncontrolled cell reproduction of NETs and duration of the align their components, which are considered biomarkers of the disease.Fluorescence microscopy allows to capture image of NETs showing the dispersion of DNA in association with protein such as LL37, which co-localized with microorganisms helping to understand how they are appearing. To begin, perform vena puncture to collect 10 milliliters of peripheral blood in tubes containing dipotassium EDTA as anticoagulant. Then perform standard blood biometry and C-reactive protein test to rule out infection or inflammation to ensure the quality of the sample.Centrifuge the peripheral blood sample to remove the platelet-rich plasma, followed by a second centrifugation and discard the remaining plasma. Dilute the resulting erythrocyte and leukocyte package in one-to-one volume ratio with one 1X DPBS. Then, in a sterile 10 milliliter glass tube, first deposit one milliliter of 1.08 grams per milliliter density solution followed by one milliliter of 1.079 grams per milliliter density solution.Then, add four milliliters of the diluted erythrocyte and leukocyte package by pouring over the walls. Centrifuge without acceleration or deceleration to avoid disturbing the gradient. Aspirate the phase corresponding to granulocytes and transfer to another sterile 10 milliliter glass tube.Wash with four milliliters of 1X DPBS at 300 G for 10 minutes at four degrees Celsius and discard the supernatant. To remove the remaining erythrocytes, treat the cells with osmotic shock by adding four milliliters of 0.2%saline solution. Incubate for two minutes at four degrees Celsius and centrifuge.Discard the supernatant and add four milliliters of 0.65%saline solution. Incubate for five minutes at four degrees Celsius to restore membrane integrity and repeat centrifugation. Remove the supernatant and re-suspend the cells in four milliliters of 1X DPBS to remove cellular debris.Again, centrifuge and re-suspend the cell pellet in two milliliters of cold HBSS buffer. To perform a trypan blue exclusion test, dilute five microliters of the cell suspension in 20 microliters of 0.4%trypan blue. Count the cells in a new bower chamber and determine cell viability using an exclusion test.Then, mount 5 microliters of the cell suspension on a slide and stain with Wright's stain for 15 seconds. Immediately fix the sample, wash with distilled water, and observe the morphology under an optical microscope. Next, add one times 10 to the fifth cells to flow cytometry tubes and stain with one microliter of 7AAD in 100 microliters of FACS buffer for 15 minutes at four degrees Celsius in the dark.Wash with 500 microliters of FACS buffer at 300 G for 10 minutes. Fix the cells with 500 microliters of 2%paraformaldehyde and store at four degrees Celsius until flow cytometry analysis. For a dead cell control, fix the cells with 200 microliters of 4%paraformaldehyde for 30 minutes and wash with 500 microliters of 1XPBS at 300 G for 10 minutes at four degrees Celsius.Discard the supernatant and add 200 microliters of 0.1%Triton X-100. Incubate for one hour at four degrees Celsius. Wash with 500 microliters of 1X PBS and stain with 7AAD.Analyze the captured data in the flow cytometer software. Load the files and double click on the unstained neutrophil file. Choose forward scatter or FSC on the X-axis and side scatter or SSC on the Y-axis to display the cell population corresponding to neutrophils.Then select the channel B3-A:PerCP vio 700-A on the X-axis to de-limit the autofluorescence of the cells and choose histogram on the Y-axis. Then, open the stained neutrophil file. Choose FSC on the X-axis and SSC on the Y-axis to display the cell population corresponding to neutrophils.Again, select the channel B3-A:PerCP vio 700-A to delimit the population of neutrophils positive for the dye 7AAD. Generate the final histogram by right-clicking on the window and selecting copy to layout editor. Add bacterial or fungal pseudohyphae in 1.5 milliliter micro tubes.Add 200 microliters, so five micromolar CFSC in 1X PBS. Mix and incubate at 37 degrees Celsius for 10 minutes in the dark. Stop the reaction by adding 500 microliters of decomplemented plasma and centrifuge.Discard the supernatant and wash the pellet with one milliliter of 1XPBS with centrifugation. Then re-suspend the microorganisms in 250 microliters of 1X PBS. Prepare 50 microliter aliquots in 1.5 milliliter microtubes with two times 10 to the seventh bacteria or two times 10 to the fifth pseudohyphae for NET induction.Place 10 by 10 millimeters sterile glass cover slips in a 24-well plate. Cover with 10 microliters of 0.001%poly l-lysine for one hour at room temperature and wash twice with 100 microliters of 1X PBS. Air dry and irradiate with UV light for 15 minutes.Next, replace HBSS solution of the neutrophil suspension prepared earlier with RPMI 1640 medium supplemented with 10%heat decomplemented autologous plasma. Add 350 microliters of this cell suspension to the 24-well plate for a final concentration of two times 10 to the fifth neutrophils per well. Incubate the plate for 20 minutes at 37 degrees Celsius with 5%carbon dioxide to allow the cells to adhere to the bottom of the wells.To induce net formation, add the bacteria stimuli MOI100 and pseudohyphae at MOI1. Also add the biochemical stimuli and prepare a control without a stimulus by adding 50 microliters of HBSS. Obtain a final volume of 400 microliters per well and mix on a plate shaker at 140 RPM for 30 seconds.Then incubate for four hours at 37 degrees Celsius and 5%carbon dioxide. After net induction, remove the supernatant from the wells by pipetting carefully and fix the cells with 300 microliters of 4%paraformaldehyde for 30 minutes. Wash the cells with 200 microliters of 1X PBS without centrifuging and add 200 microliters of blocking buffer for 30 minutes.For LL37 stain, permeabilize the cells with 200 microliters of 0.2%Triton X-100 in 1X PBS for 10 minutes and wash with 1X PBS twice. Mount the cover slips on glass slides. DNA stain the cells with two microliters of DAPI and store at minus 20 degree Celsius until analysis by confocal fluorescence microscopy.The dynamic cellular phases were visualized after double density gradient purification. The typical neutrophil morphology was confirmed by right staining. The cells also showed optimal viability observed by trypan blue exclusion without membrane disruption.Analysis of SSC versus FSC by flow cytometry confirmed a population corresponding to PMN with high cell purity. PMN dot plots for unstained cells versus 7AAD stain cells and their respective histograms indicated a high cell viability in the freshly isolated neutrophils compared to the permeabilized control cells. After stimulating the neutrophils with chemical and microbial stimulants, NETs were visualized by fluorescence microscopy using DNA DPI and anti-LL37.The microorganisms were pre-stained with CFSE. PMA-induced suicidal net formation indicated by co-localization with LL37. Whereas NETs formed with HOCI showed minor DNA dispersion in the extracellular space that corresponded to vital NET formation.NET induced by fungal pseudohyphae showed low concentrations of DNA released into the extracellular space with filamentous structures characteristic of the vital NET formation. S.aureus showed vital NET formation whereas P.aeruginosa induced the release of large concentrations of DNA with a cloudy morphology that co-localized with LL37 indicating suicide NET formation. Performing double density gradient methodology allows us to obtain a high purity and viability of neutrophils.And we elevate the washings, we avoid damaging the structure of them. Following this method we can analyze the effect of substances or cells in NET production as well as if they are involved in some mechanism or their use as therapies in autoimmune disease.

morphological-compositional-analysis-neutrophil-extracellular-traps

Research

JoVE Journal

Immunology and Infection

2.2K Views.  Universidad Autónoma “Benito Juárez” de Oaxaca. This protocol demonstrates the heterogeneity in composition, structure, and morphology of neutrophil extracellular traps, depending on their inducing stimulus under comparable in vitro conditions in human neutrophils from healthy individuals. High neutrophil purity and viability are obtained. That allows to compare the concentration of DNA, LL-37, and enzyme activity of myeloperoxidase, elastase, and cathepsin G, released by neutrophil extracellular traps.This protocol allowed to analy...