Name: High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro (Video) | JoVE Journal
Uploaded: 2016-06-18T13:00:00.000+00:00
Duration: 11 min 3 s
Description: Learn high throughput assays to quantitate neutrophil extracellular traps (NETs) released from human neutrophils in vitro. These methods enable researchers to study inflammation mechanisms and identify therapeutic targets for inflammatory diseases.

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5 minutes

Special thanks to the personnel of the University of Georgia Health Center laboratory for their continuous support of our work on isolating human neutrophils. This work was supported by the start-up fund of Dr. Rada provided by UGA Office of Vice President for Research.

The Institutional Review Board of the University of Georgia approved the human subject study to collect peripheral blood from healthy volunteers (UGA# 2012-10769-06).5,7,8 Volunteers signed the required informed consent form before blood draw. The research performed in this article is in compliance with the ethical guidelines for medical research involving human subjects of the Declaration of Helsinki.
1. Isolation of Neutrophils from Peripheral Human Blood (Figure 1)
Note: There are several ways to isolate neutrophils from peripheral blood. The following protocol provides one possibility. This protocol yields large numbers of non-activated human neutrophils capable of releasing NETs upon external stimulation. Use 40 ml whole blood obtained from healthy volunteers by venipuncture.7,9
<ol>
	<li>Aliquot 20 ml blood into two 50 ml conical tubes. Add 10 ml 6% Dextran. Mix gently.</li>
	<li>After 20 min transfer the leukocyte-rich upper phase without taking any pelleted red blood cells into clean 50 ml conical tubes, and fill it up with sterile PBS.</li>
	<li>Centrifuge at 400 x g 10 min. Resuspend pellet in 4 ml sterile PBS.</li>
	<li>Prepare a 5-step Percoll gradient of 85%-80%-75%-70%-65% in two 15 ml conical tubes and layer 2 ml leukocyte suspension on top of each gradient.</li>
	<li>Centrifuge 800 x g for 30 min with brakes off.</li>
	<li>Collect all the cells (neutrophils) in the layers at the 75%/80% and 70%/75% interface.</li>
	<li>Wash cells twice in PBS (350 x g, 10 min, RT) and count cells using a hemocytometer.</li>
</ol>
2. Measurement of Kinetics of Extracellular DNA Release Using a Microplate Fluorimeter
Note: This method enables measurement of changes in fluorescence of a membrane-impermeable DNA-binding dye indicative of DNA release on a 96-well microplate format (Figure 2).
<ol>
	<li>Prepare a suspension of neutrophils in assay medium (HBSS + 1% (v/v) autologous serum + 5 mM glucose) at a concentration of 2 x 106 cells/ml.</li>
	<li>Add 5 &#181;mol/L of the membrane impermeable DNA-binding dye. Mix gently.</li>
	<li>Aliquot human neutrophils (50 &#181;l/well) onto 96-well black, transparent bottom microplates. Make sure the assay medium covers the entire bottom of the well. If not, tap the plate gently on the side.</li>
	<li>Warm up the plate containing neutrophils for 10 min at 37 &#176;C.</li>
	<li>In the meantime prepare solutions of stimuli at double of their final concentrations to be used (in 37 &#176;C warm assay medium). As a positive NET control use human neutrophils with 100 nM phorbol-myristate-acetate (PMA) for 4 hr to trigger maximal NET release.5,8 PMA stimulation for 4 hr ensures maximal NET release while spontaneous NET formation remains low5,8.</li>
	<li>Prepare the microplate fluorimeter for the measurement (4 hr, 37 &#176;C, 530 nm for excitation and 590 nm for emission).</li>
	<li>Stimulate neutrophils by adding 50 &#181;l stimulus solution to 50 &#181;l neutrophil suspensions. Use 0.5 &#181;g/ml saponin in one well to measure maximal DNA release signal.</li>
	<li>Place plate in reader and measure changes in fluorescence for 4 hr at every 2 min with no shaking.</li>
	<li>For data analysis, calculate normalized increases in fluorescence over time.
	<ol>
		<li>Subtract baseline fluorescence from endpoint values for all wells including saponin (Figure 2A, B). Rise in saponin fluorescence should be the highest signal. It is referred to as &#34;maximal DNA release&#34; (Figure 2A).</li>
		<li>Calculate percentages of DNA release in unknown samples by dividing increases in fluorescence of unknown samples by maximal DNA release.</li>
		<li>Average results of replicates and present them as &#34;% of maximal DNA release&#34; (Figure 2C).</li>
	</ol>
	</li>
</ol>
3. Quantitation of NET Formation by MPO-DNA and HNE-DNA ELISA Assays
Note: These assays modified (MPO-DNA) or established (HNE-DNA) in our laboratory quantitate NET formation by measuring levels of MPO-DNA and HNE-DNA complexes.5
<ol>
	<li>Coat 96-well high-binding capacity ELISA plates overnight with capture antibodies (50 &#956;l/well): anti-MPO (1:2,000 dilution) or anti-HNE (1:2,000).</li>
	<li>Wash ELISA plates three times with PBS (200 &#956;l/well) and block them with 5% BSA and 0.1% human serum albumin for 2 hr at RT (200 &#956;l/well). Wash three times with PBS.</li>
	<li>Seed neutrophils on 96-well microplates at a density of 100,000 cells/well in 100 &#181;l assay medium, and stimulate NET formation. Incubate cells: 4 hr for 37 &#176;C.</li>
	<li>Perform a limited DNase digestion by adding 2 U/ml DNase to neutrophil supernatants, mix them well. Keep them at RT.</li>
	<li>Stop the DNase after 15 min by adding 11 &#181;l 25 mM EGTA (final concentration 2.5 mM). Mix well. Collect supernatants in clean microfuge tubes. Spin samples to get rid of cell debris at 300 x g for 5 min at RT. Transfer their supernatants into clean microfuge tubes. Keep them on ice until ready.</li>
	<li>Use an aliquot of a previously prepared &#34;NET standard&#34;. 
	Note: The NET-standard is a mix of DNase-digested supernatants of PMA-stimulated neutrophils obtained from at least 5 different, independent human donors.
	<ol>
		<li>To use the same standard for a long time, collect and aliquot a large volume of neutrophil supernatants from each individual donor. Collecting for example 2 ml of DNase-digested supernatants of PMA-stimulated neutrophils will result in 200 aliquots (10 &#181;l each) enough for 200 ELISA plates. Data characterizing the NET standard are found in Figure 5.</li>
		<li>Stimulate human neutrophils with 100 nM PMA for 4 hr and apply DNase digest to their supernatants according steps 3.4-3.5.</li>
		<li>Collect, pool, aliquot (10 &#181;l) and freeze (-20 &#176;C) neutrophil supernatants.</li>
		<li>To prepare the &#34;NET-standard&#34;, thaw one aliquot/donor obtained from at least five separate donors, mix them and keep them on ice.</li>
		<li>Discard thawed samples and use fresh ones for the next experiment.</li>
		<li>Prepare a 1:2 serial dilution of the NET-standard in PBS+EGTA.</li>
	</ol>
	</li>
	<li>Dilute DNase1-digested samples (standard and unknown supernatants) 20-fold in PBS+EGTA and aliquot them on ELISA plates coated with capture antibodies.</li>
	<li>Incubate ELISA plates over night at 4 &#176;C and wash them three times with PBS.</li>
	<li>Apply detection antibody solution (100 &#181;l/well): horse radish peroxidase conjugated anti-DNA antibody (1:500, mouse). Incubate for 1 hr in dark.</li>
	<li>After four washes with PBS add TMB peroxidase substrate: 100 &#181;l/well, 30 min. Blue coloration is an indication for peroxidase activity (NETs present).</li>
	<li>Stop reaction by adding 100 &#956;l/well 1 M HCl. The blue solutions will turn yellow (Figure 5A).</li>
	<li>Read absorbance at 450 nm using a microplate photometer.</li>
	<li>For data analysis, calculate the amount of MPO-DNA or HNE-DNA complexes compared to the NET standard.
	<ol>
		<li>Subtract the background optical density value of the assay medium from all samples.</li>
		<li>Plot the absorbance values (X-axis) of the diluted standard samples against their relative NET content (Y-axis) (Figure 5A).</li>
		<li>Using the nonsaturated range of this standard curve establish a trend line (use best exponential fit from the software used) (Figure 5A). The equation of this trend line determines the conversion of OD values to quantitative amounts of NETs.</li>
		<li>Insert the measured OD values of the unknowns into the trend-line equation from 3.13.3 that will give the amount of NETs in the unknown sample as percentage of the NET-standard (Figure 5A).</li>
		<li>Calculate averages of replicates (triplicates) and present the final data as &#34;amount of MPO-DNA or HNE-DNA complexes (% of standard)&#34; (Figure 5C).</li>
	</ol>
	</li>
</ol>

<ol><li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+kill+bacteria%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neutrophil extracellular traps kill bacteria.</a> Science. 303, 1532-1535 (2004).</li>
<li>Fuchs, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+cell+death+program+leads+to+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Novel cell death program leads to neutrophil extracellular traps.</a> J Cell Biol. 176, 231-241 (2007).</li>
<li>Kessenbrock, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Netting+neutrophils+in+autoimmune+small-vessel+vasculitis%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Netting neutrophils in autoimmune small-vessel vasculitis.</a> Nat Med. 15, 623-625 (2009).</li>
<li>Leffler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Degradation+of+neutrophil+extracellular+traps+co-varies+with+disease+activity+in+patients+with+systemic+lupus+erythematosus%5BTitle%5D)+AND+%22Arthritis+Res+Ther%22%5BJournal%5D)">Degradation of neutrophil extracellular traps co-varies with disease activity in patients with systemic lupus erythematosus.</a> Arthritis Res Ther. 15, R34(2013).</li>
<li>Yoo, D. G., Floyd, M., Winn, M., Moskowitz, S. M., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NET+formation+induced+by+Pseudomonas+aeruginosa+cystic+fibrosis+isolates+measured+as+release+of+myeloperoxidase-DNA+and+neutrophil+elastase-DNA+complexes%5BTitle%5D)+AND+%22Immunol+Lett%22%5BJournal%5D)">NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.</a> Immunol Lett. 160, 186-194 (2014).</li>
<li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+hypercitrullination+mediates+chromatin+decondensation+and+neutrophil+extracellular+trap+formation%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.</a> J Cell Biol. 184, 205-213 (2009).</li>
<li>Pang, L., Hayes, C. P., Buac, K., Yoo, D. G., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudogout-associated+inflammatory+calcium+pyrophosphate+dihydrate+microcrystals+induce+formation+of+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps.</a> J Immunol. 190, 6488-6500 (2013).</li>
<li>Yoo, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Release+of+cystic+fibrosis+airway+inflammatory+markers+from+Pseudomonas+aeruginosa-stimulated+human+neutrophils+involves+NADPH+oxidase-dependent+extracellular+DNA+trap+formation%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation.</a> J. Immunol. 192, 4728-4738 (2014).</li>
<li>Brinkmann, V., Laube, B., Abu Abed,, Goosmann, U., Zychlinsky, C., A, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps%2C+how+to+generate+and+visualize+them%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Neutrophil extracellular traps, how to generate and visualize them.</a> J. Vis. Exp. , (2010).</li>
<li>Rada, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pyocyanin-enhanced+neutrophil+extracellular+trap+formation+requires+the+NADPH+oxidase%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase.</a> PLoS One. 8, e54205(2013).</li>
<li>Neumann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+antimicrobial+peptide+LL-37+facilitates+the+formation+of+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps.</a> Biochem J. 464, 3-11 (2014).</li>
<li>Leon, S. A., Shapiro, B., Sklaroff, D. M., Yaros, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Free+DNA+in+the+serum+of+cancer+patients+and+the+effect+of+therapy%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Free DNA in the serum of cancer patients and the effect of therapy.</a> Cancer Res. 37, 646-650 (1977).</li>
<li>Raptis, L., Menard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitation+and+characterization+of+plasma+DNA+in+normals+and+patients+with+systemic+lupus+erythematosus%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Quantitation and characterization of plasma DNA in normals and patients with systemic lupus erythematosus.</a> J Clin Invest. 66, 1391-1399 (1980).</li>
<li>Chang, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elevated+cell-free+serum+DNA+detected+in+patients+with+myocardial+infarction%5BTitle%5D)+AND+%22Clin+Chim+Acta%22%5BJournal%5D)">Elevated cell-free serum DNA detected in patients with myocardial infarction.</a> Clin Chim Acta. 327, 95-101 (2003).</li>
</ol>

The authors have nothing to disclose.

NETs represent a fascinating novel mechanism by which neutrophils kill pathogens.1 Although the literature of NETs has been continuously expanding over the last ten years since their discovery, several important questions related to their role in biology, mechanism and regulation remain unclear. Appropriate methodology has to be developed to measure NETs, this very unique antimicrobial mechanism. This article describes methods that can be used to quantitate NETs in a high throughput manner. The first assay fol...

NET formation is a novel mechanism by which neutrophils fight pathogens.1 The core of NETs is nuclear DNA.1 This DNA network is associated with neutrophil granule proteins and histones.1 The main form of NET formation requires the death of neutrophils characterized by chromatin decondensation, disappearance of granular and nuclear membranes, translocation of neutrophils elastase to the nucleus, citrullination of histones and finally the spill of DNA-based NETs.2 NETs entrap and kill a wide variety of microbes and are an essential part of the innate immune weapon repertoire. Uncontrolled NET formation has, however, been linked to numerous autoinflammatory diseases.3,4 Despite their increasingly established relevance, little is known about the mechanism and regulation of NET release. 
Neutrophils dying by releasing NETs are different from apoptotic or necrotic neutrophils.3,5 NET-releasing neutrophils show several features that are characteristic for NET formation. Granule components are associated with DNA in NETs.1 Myeloperoxidase (MPO) and human neutrophil elastase (HNE) are both found in primary granules in resting cells but are translocated to the nucleus to bind to DNA in NETs.1 MPO-DNA and HNE-DNA complexes are specific for NETs, do not occur in apoptotic or necrotic neutrophils.1,3,5 Chromatin decondensation is another feature typical for NETosis.2 NET release also requires citrullination of histones by peptidyl aminidase 4 (PAD4).6 Citrullinated histones are hallmarks of neutrophils that underwent NET release.6
Here three methods are introduced that in combination provide excellent tools to quantitate NETs on a high-throughput scale. The first assay has been used on the field with different changes and quantitates extracellular DNA release in a microplate format. The second and third assays provide confirmation of NETs by measuring NET-specific MPO-DNA and HNE-DNA complexes.

<table><tbody><tr><td>Anti-Human Neutrophil Elastase&amp;nbsp; Rabbit Ab&amp;nbsp;</td><td>Calbiochem</td><td>481001</td><td>1:2,000x coated</td></tr><tr><td>Anti-Myeloperoxidase Ab (Rabbit)</td><td>Millipore</td><td>07-496</td><td>1:2,000x coated</td></tr><tr><td>DNase-1</td><td>Roche</td><td>10-104-159-001</td><td>1 &amp;#181;g/ml used for digestion</td></tr><tr><td>20 mM EGTA/ PBS</td><td>Sigma-Aldrich</td><td>E3889-25G</td><td></td></tr><tr><td>2.5 mM EGTA/PBS</td><td>Sigma-Aldrich</td><td>E3889-25G</td><td></td></tr><tr><td>Cell death detection ELISA Anti-DNA POD</td><td>Roche</td><td>11544675001</td><td>1:500x&amp;nbsp;</td></tr><tr><td>Eon Microplate Spectrophotometer</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 All-in-One microplate software</td><td>Biotek</td><td></td><td>analytical tool (ELISA)</td></tr><tr><td>Sytox orange</td><td>Life Technology</td><td>S11368</td><td>0.2% final concentration/volume</td></tr><tr><td>1 M Hepes</td><td>Cellgro</td><td>25-060-Cl</td><td>Use 10 mM final concentration.</td></tr><tr><td>1 M glucose</td><td>Sigma</td><td></td><td>Use 5 mM final concentration.</td></tr><tr><td>HBSS</td><td>Corning</td><td>21-023-CM</td><td></td></tr><tr><td>Varioskan Flash Ver.2.4.3</td><td>Thermoscientific</td><td></td><td></td></tr><tr><td>PMA</td><td>Sigma</td><td>P 8139</td><td>100 nM final used</td></tr><tr><td>ELISA Plate</td><td>Greiner bio-one</td><td>655061</td><td></td></tr><tr><td>Conical tubes 15 ml</td><td>Thermoscientific</td><td>339650</td><td></td></tr><tr><td>Conical tubes 50 ml</td><td>Thermoscientific</td><td>339652</td><td></td></tr><tr><td>Percoll (pH 8.5-9.5)&amp;nbsp;</td><td>Sigma</td><td>P 1644</td><td>Sodium Chloride, Sigma, S7653-250G</td></tr><tr><td>Dextran</td><td>Spectrum</td><td>D1004</td><td></td></tr><tr><td>RPMI 1640 media</td><td>Corning Cellgro</td><td>17-105-CV</td><td></td></tr><tr><td>96 well assay plate black plate clear bottom</td><td>Costar</td><td>3603</td><td></td></tr></tbody></table>

high throughput measurement of extracellular dna release and quantitative net formation in human neutrophils in vitro

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.

The figures in this manuscript describe the method of neutrophil isolation, experimental procedures, and present representative results with explanation of data analysis. Figure 1 shows the sequential steps of human neutrophil preparation. This protocol represents only one possible way of neutrophil isolation. It yields large amounts of resting neutrophils capable of releasing NETs upon stimulation. Figure 2 shows how the fluorescence-based DNA release as...

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 ...

The overall goal of this procedure is to quantitate neutrophil extracellular traps, or NETs, released from human neutrophils on a high throughput scale. The methods presented here can help answer key questions in the fields of inflammation and altering inflammatory diseases through the study of the mechanism and regulation of in vitro neutrophil extracellular trap formation in human neutrophils. The implications of this technique involves identification of a central NET mediating signaling pathways and NET inhibitory or NET stimulating molecules.To isolate neutrophils from blood, aliquot 20 milliliters of human blood into 250 milliliter conical tubes. Add 10 milliliters of 6%dextran, then gently mix and allow the solutions to sit at room temperature for 20 minutes. Carefully transfer the leukocyte rich upper phase into clean 50 milliliter conical tubes, and use sterile PBS to fill the tube to the top.Then centrifuge the sample at 400 times G for ten minutes and use 15 milliliters of sterile PBS to resuspend the pellet. Repeat this wash step one more time. Next, prepare five step percoll gradients of 85, 80, 75, 70 and 65 percent in 215 milliliter conical tubes and layer two milliliters of the leukocyte suspension on top of each gradient.Then, centrifuge the gradients at 800 times G for 30 minutes with the break off. After centrifugation, collect the cells in the layers at the 75-80%and 70-75%interfaces. Use PBS to wash the cells twice, and then use a hemocytometer to count the cells.To measure the kinetics of extracellular DNA release, prepare a suspension of neutrophils in assay medium at a concentration of two times ten to the six cells per milliliter. Add five micromoles per liter of SYTOX orange, which is a membrane-impermeable DNA binding dye and gently mix. Aliquot human neutrophils into sterile 96-well black transparent bottom microplates ensuring that the assay medium covers the entire bottom of the well.If not, gently tap the plate on the side. Warm the plate at 37 degrees celsius for 10 minutes. In the mean time, use 37 degrees celsius assay medium to prepare solutions containing stimuli at double their final concentrations to be used.As a positive NET control, prepare 100 nanomolar phorbol myristate acetate, or PMA, to treat human neutrophils for four hours to trigger maximal NET release. Next, prepare the microplate fluorometer for measurement. Stimulate neutrophils by adding 50 microliters of stimulus solution to 50 microliters of neutrophils suspensions.Add a final concentration of 0.5 milligrams per milliliter of saponin to one well, to measure the maximal DNA release signal. Place the plate in the reader and measure changes in fluorescence at two minute intervals for four hours without shaking. To analyze the data, calculate normalized increases in fluorescence over time by subtracting the baseline fluorescence from end point values for all wells including saponin.The rise in saponin fluorescence should be the highest signal and is referred to as the maximal DNA release. Average the results of replicates and present them as percent of maximal DNA release. To quantitate NET formation using MPO-DNA and HNE-DNA ELISA assays, add anti-MPO or anti-HNE capture antibodies to 96-well high binding capacity ELISA plates, and incubate them overnight to coat.The next morning use 200 microliters per well of PBS to wash the ELISA plates three times. Then use 200 microliters of 5%bovine serum albumin, or BSA, and 0.1%human serum albumin, or HSA, to block the wells at room temperature for two hours. In the mean time, seed neutrophils in 100 microliters of assay medium on a 96-well plate at a density of 100, 000 cells per well and induce NET formation after addition of stimuli by incubating at 37 degrees celsius for four hours.Next, perform a limited DNase digestion by adding two units per milliliter of DNAse to the neutrophils supernatants then mix well and incubate at room temperature for 15 minutes. Then, stop the reaction by adding 15 microliters of 25 millimolar EGTA and mix well. Collect the supernatants and clean microfuge tubes and spin the samples to get rid of cell debris at 300 times G and room temperature for five minutes.Collecting NETs without limited digestion results in inconsistent data. The limited DNA digestion step is critical to obtain reproducible results. Therefore, the concentration of DNase I enzyme and the length of digestion has been optimized.Transfer the supernatant to clean microfuge tubes and keep on ice. Then, using an aliquot of previously prepared NET-standard, prepare a one to two dilution in PBS and EGTA. Now, use PBS with EGTA to dilute DNase I digested samples twenty fold and aliquot them on ELISA plates coated with capture antibodies.Incubate the plates over night at four degrees celsius. Before using PBS to wash the plates three times, apply 100 microliters per well of a detection antibody solution such as horseradish peroxidase conjugated anti DNA antibody, and incubate in the dark for one hour. After four washes with PBS, add 100 microliters per well of TMB peroxidase substrate, and incubate for 30 minutes.Blue coloration indicates peroxidase activity and the presence of NETs. To stop the reaction, add 100 microliters of one molar HCL. The blue solutions will turn yellow.Using a microplate photometer, read the absorbance at 450 nanometers. To compare the amount of MPO-DNA or HNE-DNA complexes measured compared to the NET-standard, subtract background optimal density from all samples and plot the absorbance values of the diluted standard samples against their relative NET content. Using the non-saturated range of this standard curve, establish a trend line using the best exponential fit from the software used.Insert the measured OD values of the unknowns into the trend line equation that will give the amount of NETs in the unknown sample as percent of the NET-standard. Finally, calculate the averages of replicates and present the final data as the amount of MPO-DNA or HNE-DNA complexes. Shown here is representative data from a fluorescence based DNA release assay of human neutrophils left un-stimulated or activated with PMA or P-aeruginosa.P-aeruginosa triggers NET formation in human PMNs. This graph illustrates that the averages of increase in fluorescence of replicate wells are calculated, normalized on maximal fluoresence and are expressed as DNA release. These fluorescence images represent neutrophils left untreated or stimulated with PMA.Shown here are immunofluorescence images of nonstimulated, PMA, and PA01 activated human neutrophils from an MPO-DNA and HNE-DNA Elisa assay. Extracellular DNA shows typical NET morphology and colocalizes with MPO and citrullinated histone H4, a marker of NET formation. This experiment shows MPO-DNA and HNE-DNA release in human neutrophils using the same assay.The NET-standard on average contains 19.5 micrograms per milliliter of extracellular DNA, 399 nanograms per milliliter of MPO, and 103.4 nano-grams per milliliter of HNE. Based on the molecular weights of MPO and HNE, and that one microgram of DNA contains 9.91 times 10 to the 14 nucleotides, the NET-standard contains 109 HNE molecules and 147 MPO molecules per 1, 000 nucleotides. Finally, two fold serial dilution experiments of the NET-standard reveal that the dynamic ranges of both the MPO-DNA and HNE-DNA ELISA assays are between 19 and 609 nanograms per milliliter of DNA.Once mastered, the SYTOX orange based acid can be easily and quickly assembled in one hour, leading to results the next day. The NET quantitating assay are also easy to perform and will result in data the following day if performed properly. After watching this video, you should have a good understanding of how to quantitate naturalase in human neutrophils in a high throughput manner.

high-throughput-measurement-extracellular-dna-release-quantitative

Research

JoVE Journal

Immunology and Infection

This video demonstrates an in vitro technique to generate and isolate neutrophil extracellular traps (NETs). Neutrophils are isolated from peripheral blood and stimulated with phorbol myristate acetate (PMA) to produce NETs. During incubation, the neutrophils undergo a process of cell death (NETosis) to release NETs comprising decondensed chromatin mixed with granular cytoplasmic proteins.

An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

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.

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

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.

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

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

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.

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

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.

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

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.

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

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

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

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

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

Learn high throughput assays to quantitate neutrophil extracellular traps (NETs) released from human neutrophils in vitro. These methods enable researchers to study inflammation mechanisms and identify therapeutic targets for inflammatory diseases.

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

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