The authors would like to acknowledge Dr. Paul Kubes for his guidance and mentoring that were central in the construction of this work.

NOTE: All experiments were conducted in accordance with local institutional ethical guidelines.
1. Blood Drawing
<ol>
	<li>Through antecubital venipucture, collect 14 tubes of blood from a healthy volunteer into green top (heparinized) tubes and invert each tube before settling on ice. Collect blood within 10 - 15 min of experiment to ensure optimal neutrophil yield.</li>
</ol>
2. Neutrophil Isolation from Whole Blood
<ol>
	<li>Wash each green top tube of blood with 5 ml of PBS without Ca and Mg to dilute the blood. In each of 4 x 50 ml&#160;conical tubes, add 15 ml of Lymphocyte Separation Media (LSM) at RT. Using a 18 G needle mounted on a 60 ml&#160;syringe, carefully layer the diluted blood onto the LSM, creating a sharp LSM-blood interface. 
	NOTE: Avoid mixing of the layers as much as possible.</li>
	<li>Centrifuge at 800 x g for 30 min at 21 &#176;C without break. 
	NOTE: Sudden breaks can cause mixing of the different layers.</li>
	<li>Observe the erythrocytes and neutrophils sediment at the bottom. Aspirate and discard the top 2 layers (plasma and LSM) making sure to get rid of the interface between these layers which contains lymphocytes and mononuclear cells, leaving only the bottom red layer.</li>
	<li>To each tube, add 20 ml of Phosphate Buffer Saline (PBS) and 20 ml of 6% Dextran solution. Gently invert each tube to mix with the layer of erythrocytes and neutrophils and allow to sit at RT for 30 min. 
	NOTE: This will allow red blood cells (RBCs) to sediment at the bottom.</li>
	<li>After 30 min, transfer the neutrophil rich supernatant into fresh tubes and discard the RBC pellets. Centrifuge supernatants at 450 x g at 4 &#176;C for 5 min. After centrifugation, discard supernatant. The pellet will contain mostly neutrophils and few RBCs.</li>
	<li>Prepare a lysing solution by adding 0.5 ml of lysing buffer to 4.5 ml of sterile water. Lyse remaining RBC with the prepared 5 ml of lysing solution, transferring all resuspended pellets into one tube. Allow lysis at RT in the dark for 10 min.</li>
	<li>Centrifuge at 450 x g at 4 &#176;C for 5 min. Discard supernatant and wash pellet with 5 ml PBS without Ca and Mg and centrifuge again at 450 x g at 4 &#176;C to get rid of any remaining lysing solution. The pellet obtained at this point contains the neutrophils. Resuspend the pellet in 30 ml of cold 3% RPMI and put on ice. 
	NOTE: 3% RPMI is made by supplementing RPMI media with 3% Fetal Bovine Serum (FBS)</li>
	<li>Verify purity of the sample using Methylene blue staining. &#62;95% of cells should be granulocytes with multi-lobar nuclei. Use Trypan blue staining to verify &#62;95% viability of cells and count the final neutrophil yield. Dilute neutrophils in ice cold 3% RPMI to obtain a final concentration of 5 x 106 neutrophils/ml.</li>
</ol>
3. NETs Generation
<ol>
	<li>Stimulate neutrophils with 500 nM of PMA (per 30 ml of neutrophil solution) and incubate on a 150 x 25mm flat tissue culture dish with 20 mm grid for 4 hr at 37 &#176;C 5% CO2. This will allow NETosis.</li>
	<li>After 4 hr of stimulation, gently aspirate and discard the media, leaving the layer of NETs and neutrophils adhered at the bottom. Do not disrupt this layer.</li>
	<li>Using a total of 15 ml of cold PBS without Ca and Mg per dish, wash the bottom of each dish by pipetting 15 ml of PBS on the bottom of the dish in order to lift off all adherent material from the bottom.</li>
	<li>Collect solution obtained from washing each dish (step 3.3) in a 15 ml conical tube and centrifuge for 10 min at 450 x g at 4 &#176;C. Neutrophils and any remaining cells will pellet at the bottom, leaving a cell-free NET-rich supernatant.</li>
	<li>Divide supernatant into 1.5 ml micro-centrifuge tubes and spin for 10 min at 18,000 x g at 4 &#176;C. This will allow all DNA to pellet. 
	NOTE: Centrifugation in larger tubes is easier and less time consuming if such high speeds are attainable on the regular centrifuge, otherwise will need to divide supernatants in smaller tubes in order to use high speed micro-centrifuge.</li>
	<li>Discard supernatant and resuspend all pellets obtained together in ice cold PBS to a concentration corresponding to 2 x 107 neutrophils per 100 &#956;l of PBS. This will yield the cell-free NET stock that can be used for subsequent experiments.</li>
	<li>Measure DNA concentration in the sample obtained using spectrophotometry or alternate DNA quantification tool. An adequate concentration in the sample should range between 140 - 180 ng/&#956;l.</li>
</ol>
4. NET-Cancer Cell Static Adhesion Assay
<ol>
	<li>Add 100 &#956;l of the previously obtained NET stock per well in a 96 well flat bottom plate and allow to coat wells O/N at 4 &#176;C in the dark.</li>
	<li>Between 12 and 20 hr&#160;later, verify formation of a uniform monolayer of cell free NETs at the bottom of the wells under the microscope (Figure 1). At this point, gently aspirate all non-adherent material out of the wells, making sure not to disrupt the NET monolayer at the bottom.</li>
	<li>Add 100 &#956;l of 1% Bovine Serum Albumin (BSA) blocking solution to each well and leave for 1 h at RT.</li>
	<li>Detach A549 cancer cells from a T-75 flask of using 2 ml of 0.25% Trypsin solution. Once detached, add 10 ml of A549 media to trypsinized cells and centrifuge at 450 x g at 4 &#176;C for 5 min. Discard supernatant and resuspend cells in media to obtain a concentration of 2 x104 cancer cells per 100 &#956;l media. 
	NOTE: A549 cells were grown separately. Briefly, cells were cultured and maintained in DMEM F12 media containing 10% FBS and 1% Penicillin Streptomycin and incubated at 37 &#176;C 5% CO2. Once 70 - 80% cell confluence was reached, they were detached using 0.25% Trypsin-EDTA and resuspended in the same media described above.</li>
	<li>Stain cancer cells using CFSE by adding 1 &#956;l of CFSE per ml of media and leave to stain at RT for 10 min. After staining, centrifuge down at 450 x g at 4 &#176;C for 5 min then discard supernatant and resuspend cells in initial volume of media to obtain a concentration of 2 x104 cancer cells per 100 &#956;l media.</li>
	<li>After 1 hr of NETs blocking, gently aspirate blocking solution and add 2 x104 cancer cells in media, which is equivalent to 100 &#956;l, per well over the NET monolayer and allow to adhere for 90 min at 37 &#176;C 5% CO2. Gently aspirate the cells and add 100 &#956;l of PBS to each well to wash any non-adherent cells.</li>
	<li>In some wells, add 1000U of DNAse1 per well for 10 min before washing to degrade NETs. This will serve as negative control. In other wells, add 100 &#956;l of sterile water per well for 10 min, which will serve as the vehicle control (VC). 
	NOTE: 3 replicates per condition are usually performed to increase sample size.</li>
	<li>Aspirate and discard all solution in the wells leaving only NETs and adherent cancer cells at the bottom. Add 100 &#956;l of 4% formaldehyde solution per well to fix adherent cancer cells to NETS and read assay under the fluorescence microscope (Figure 1). Plot and analyze the results (Figure 3).</li>
</ol>

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G., Jenne, C. N., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+neutrophil+extracellular+traps+capture+bacteria+from+the+bloodstream+during+sepsis.">Intravascular neutrophil extracellular traps capture bacteria from the bloodstream during sepsis.</a> Cell Host Microbe. 12 (3), 324-333 (2012).</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> J Immunol. 185 (12), 7413-7425 (2010).</li><li>Villanueva, E., 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=Netting+neutrophils+induce+endothelial+damage,+infiltrate+tissues,+and+expose+immunostimulatory+molecules+in+systemic+lupus+erythematosus.">Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus.</a> J Immunol. 187 (1), 538-552 (2011).</li><li>Khandpur, 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=NETs+are+a+source+of+citrullinated+autoantigens+and+stimulate+inflammatory+responses+in+rheumatoid+arthritis.">NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.</a> Sci Transl Med. 5 (178), 178ra140 (2013).</li><li>Demers, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancers+predispose+neutrophils+to+release+extracellular+DNA+traps+that+contribute+to+cancer-associated+thrombosis.">Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis.</a> Proc Natl Acad Sci U S A. 109 (32), 13076-13081 (2012).</li><li>Hahn, S., Giaglis, S., Hoesli, I., Hasler, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+NETs+in+reproduction:+from+infertility+to+preeclampsia+and+the+possibility+of+fetal+loss.">Neutrophil NETs in reproduction: from infertility to preeclampsia and the possibility of fetal loss.</a> Front Immunol. 3, 362 (2012).</li><li>Cools-Lartigue, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+sequester+circulating+tumor+cells+and+promote+metastasis.">Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis.</a> J Clin Invest. , (2013).</li><li>Cools-Lartigue, J., Spicer, J., Najmeh, S., Ferri, L. <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+cancer+progression.">Neutrophil extracellular traps in cancer progression.</a> Cell Mol Life Sci. , (2014).</li><li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+how+to+generate+and+visualize+them.">Neutrophil extracellular traps: how to generate and visualize them.</a> J Vis Exp. (36), (2010).</li><li>Maqbool, M., Vidyadaran, S., George, E., Ramasamy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+laboratory+procedures+for+isolating+human+peripheral+blood+derived+neutrophils.">Optimisation of laboratory procedures for isolating human peripheral blood derived neutrophils.</a> Med J Malaysia. 66 (4), 296-299 (2011).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+lymphocytes,+granulocytes+and+macrophages.">Isolation of lymphocytes, granulocytes and macrophages.</a> Scand J Immunol. Suppl. 5, 9-15 (1976).</li><li>Calado, R. T., 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=Sex+hormones,+acting+on+the+TERT+gene,+increase+telomerase+activity+in+human+primary+hematopoietic+cells.">Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells.</a> Blood. 114 (11), 2236-2243 (2009).</li><li>Zhong, C., Qu, X., Tan, M., Meng, Y. G., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+regulation+of+bv8+in+human+blood+cells.">Characterization and regulation of bv8 in human blood cells.</a> Clin Cancer Res. 15 (8), 2675-2684 (2009).</li><li>Wu, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+leukocyte+labeling+with+intravenous+ferumoxides/protamine+sulfate+complex+and+in+vitro+characterization+for+cellular+magnetic+resonance+imaging.">In vivo leukocyte labeling with intravenous ferumoxides/protamine sulfate complex and in vitro characterization for cellular magnetic resonance imaging.</a> Am J Physiol Cell Physiol. 293 (5), C1698-C1708 (2007).</li><li>Saffarzadeh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+directly+induce+epithelial+and+endothelial+cell+death:+a+predominant+role+of+histones.">Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones.</a> PLoS One. 7 (2), e32366 (2012).</li><li>Fuchs, T. 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+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> J Cell Biol. 176 (2), 231-241 (2007).</li></ol>

The authors have nothing to disclose

The Neutrophil Extracellular Trap isolation protocol demonstrated in this video combines different techniques used in the literature for neutrophil isolation and NET formation. It simplifies a fairly complex and variable process and offers a very reliable and replicable way to isolate purified cell-free NETs with fewer steps than other protocols. Furthermore, readily available reagents are employed adding to the protocols simplicity and significantly reducing its cost. The application of NETs towards a static adhesion assay serves to demonstrate the flexibility afforded by this isolation technique, as NET concentration can easily be manipulated to suit a particular goal (17). Accordingly, NETs isolated by the demonstrated technique can be used for various types of assays including dynamic adhesion assays, migration assays, proliferation assays immunofluorescence and confocal imaging, electron microscopy, flow cytometry as well as traditional Western blotting.
There are a number of steps in this protocol that are critical for its success. First, neutrophils may be accidentally activated during the process of isolation leading to apoptosis. It is therefore important to perform all isolation steps under sterile conditions under the fume hood, avoid aggressive mixing of tubes, keep all samples on ice after the RBC lysis step, and avoid long wait times between steps. Second, if the goal of the assay is to coat wells for the purpose of assessing adhesion, it is important to respect the suggested NET concentration per well as well as the proposed final DNA concentration in the &#8220;NET stock&#8221; as this will ensure an even and consistent monolayer of NETs. Lower concentration may produce only patchy coating of the well and therefore less reliable results. Finally, it is also important to handle the NET monolayer very carefully while performing the assay to prevent its disruption. This can be facilitated by adjusting the intensity of the suction and pipetting on the sides of the wells.
Limitations of this technique include the fact that it still requires a significant amount of time to produce NETs, mostly caused by the 4 hr PMA stimulation step. In addition, a significant amount of blood is required to isolate a sufficient number of neutrophils required for one experiment. There definitely is loss of a significant amount of NETs at the first centrifugation step (step 3.4.) when discarding neutrophils. Modifications to minimize NET loss at this point could significantly increase the final NET yield. Also, isolated NETs must be used within 12 - 24 hr of their isolation, which requires careful experiment planning and time management.

Neutrophil extracellular traps (NETs) are newly discovered neutrophil-derived elements composed of strands of extracellular DNA decorated by hundreds of proteins and histones. They are secreted by neutrophils in the context of infection and inflammation following specific stimuli and they were initially recognized to be part of the neutrophil&#8217;s defense mechanisms against infections. They were first shown to promote trapping of various microbial invaders including bacteria, viruses and fungi 1-3. Trapping of the microbe would then lead to its destruction both directly by NET-associated proteins 3,4 and indirectly through local recruitment of phagocytic cells 5,6. The role of NETs however does not seem to be limited to host defense. More recently, they have been shown to play an important role in autoimmune diseases 7,8, thrombosis 9, pregnancy-related disorders 10 and even cancer progression 11,12. Accordingly, it appears that NETs play an important role in a large number of diverse physiologic processes. However, given the novel nature of such research, much remains to be elucidated with respect to the mechanisms by which NETs exert their effects. Herein, we present a simplified method for the isolation of NETs in the absence of neutrophils.
In the following video, we demonstrate a simplified and easy technique to isolate NETs from human whole blood. Cell-free NETs can then be used in a number of in vitro experiments including staining, imuunofluorescence, blotting as well as a number of assays such as adhesion, proliferation and migration. Other protocols exist 13, 19, however they are usually lengthy, complex, expensive, and often, low yield 13. This simplified protocol uses basic reagents and decreases the number of steps required to isolate neutrophils, therefore minimizing the length of the procedure while maximizing yield.
The following method combines different techniques for neutrophil isolation previously described in the literature to obtain a simple protocol yielding a very pure sample of live neutrophils. As suggested in the literature, venous blood is collected in EDTA tubes and used within 10 min to avoid neutrophil activation 14. We use Lymphocyte Separation Media (LSM) density gradient centrifugation to isolate granulocytes and red blood cells from heparinized whole blood, a method adapted from the Ficoll density centrifugation technique initially described by Boyum et al 15. LSM is a modification of the Boyum formulation that substitutes sodium diatrizoate for the sodium metrizoate and has been used successfully in many studies to isolate neutrophils 16-18.
Following differential density centrifugation, monocytes and lymphocytes are discarded; red blood cells are then sedimented using a 6% Dextran solution 14 and the remaining RBCs are lysed to obtain a population of pure neutrophils, which is verified with Trypan blue and Methylene blue staining.
Numerous agents have been used to induce NETosis both in vitro and in vivo, including lipopolysaccharide (LPS), and phorbol myristate acetate (PMA) and interleukins (IL-8) 3,5,6. In the following protocol, isolated neutrophils are stimulated with 500 nM PMA for 4 hr, which has been shown to be an adequate concentration to allow consistent and reliable NET formation without promoting apoptosis 19,20.
After isolation, we demonstrate how cell-free NETs obtained from this protocol can be used in an adhesion assay. DNAse1 is used to digest and degrade NETs as previously described and thus serves as a control 2,3,11. Other options include the use of Neutrophil elastase inhibitor (NEi) to inhibit NET formation, which is an acceptable alternative when the goal is to inhibit the NET formation rather than effect their degradation 11. Although NEi has a number of varied functions, it has been previously shown that NET deposition can be inhibited by NEi through blockage of chromatin decondensation, nuclear degranulation and neutrophil death 12

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (PBS), Modified, without calcium chloride and magnesium chloride</td><td>Wisent</td><td>311-425-CL</td><td></td></tr><tr><td>Lymphocyte Separation Medium (LSM)</td><td>Wisent</td><td>305-010-CL</td><td></td></tr><tr><td>Dextran hydrochloride (powder)</td><td>Spectrum</td><td>DE130</td><td>6% Dextran solution is made by supplementing PBS with Ca and Mg with 6% Dextran powder</td></tr><tr><td>RPMI-1640 Medium with L-glutamine</td><td>Wisent</td><td>350-000-CL</td><td>3% RPMI solution is made by supplementing RPMI with 3% Fetal Bovine serum</td></tr><tr><td>BD Pharm Lyse Lysing buffer</td><td>BD Biosciences</td><td>555899</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma-Alrdrich</td><td>P8139</td><td></td></tr><tr><td>DNAse1</td><td>Roche</td><td>11284932001</td><td></td></tr><tr><td>F12 DMEM</td><td>Wisent</td><td>319-075-CL</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Wisent</td><td>080-150</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>CFSE</td><td>Invitrogen</td><td>C1157</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Integrid tissue culture dish with 20 mm grid (150 x 25 mm)</td><td>Falcon</td><td>353025</td><td></td></tr></tbody></table>

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

Successful achievement of a static adhesion assay with NETs requires adequate coating of the wells with a monolayer of NETs before the addition of cancer cells (Figure 1). All subsequent steps must be done with care not to disrupt that layer. When an adequate layer of NETs is formed, it is expected to see significant cancer cells adhesion to NETs as seen in Figure 2A&#160;and 2C. This effect is abrogated by the addition of DNAse1, which degrades NETs as seen in Figures 2B&#160;and 2D. Conversely, there should be no significant decrease in A549 adhesion to NETs in the presence of the vehicle control (Figure 3). To obtain a good estimation of mean cancer cell adhesion per high power field (hpf), it is recommended to count at least 4 random hpfs per well. Fields that are not well coated in NETs due to technical issues should not be taken into consideration for counting as these might falsify results.
<img alt="Figure 1" src="/files/ftp_upload/52687/52687fig1.jpg" /> 
Figure 1.&#160;NET Monolayer.&#160;Light microscopy images of the 96 wells plate wells coated with NETs showing (A) uniform monolayer of NETs throughout well as opposed to (B) inadequate coating of the well with an interrupted layer of NETs. Scale bars represent 40 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52687/52687fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52687/52687fig2.jpg" /> 
Figure 2.&#160;A549 Cancer Cell Adhesion to NETs In Vitro. (A) A549 cancer cells (arrows) adhere to monolayer of NETs in vitro. (B) Addition of DNAse1 decreases significantly the level of cancer cell adhesion; (C) shows a representative image of cancer cell adhesion on NETs under fluorescent light under fluorescent microscopy (Nikon TE300) before (C) and after (D) addition of DNAse1. Scale bars represent 40 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52687/52687fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52687/52687fig3.jpg" /> 
Figure 3. Results for In Vitro Adhesion Assay.&#160;GraphPad software was used to plot and analyze results from the A549 cancer cell adhesion assay to NETs. In the presence of DNAse, adhesion was 13.90% compared to untreated NETs. In the presence of vehicle control (VC), adhesion was 77.26% compared to untreated NETs. Data is presented as mean +/- SEM. Significance was determined using Kruskall Wallis test with **p&#60;0.001.

Watch this Scientific Journal Video about Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling at JoVE.com

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.

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 ...

simplified-human-neutrophil-extracellular-traps-nets-isolation

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

29.8K Views.  McGill University. 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.

Video: Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

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 ...

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

Cancer Research

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

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

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

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

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

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

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

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

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

Medicine

Staining and Imaging to Visualize Neutrophil Extracellular Traps

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 &#181;m thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.
Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

Author Spotlight: Neutrophil Extracellular Traps Imaging in Human and Mouse Tissues 

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...

Quantifying NET Formation Using Dual-Dye Live Cell Analysis

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

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Author Spotlight: Quantifying Neutrophil Extracellular Traps in Disease and Drug Screening Using Dual-Color Live-Cell Imaging

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

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

Real-Time Microscopy for Studying NETosis Kinetics and Screening Inhibitors

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

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

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

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

Biology

An In Vitro Technique to Generate Human Neutrophil Extracellular Traps

Source: Najmeh, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52687">Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.</a> J. Vis. Exp. (2015)
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.

Source: Najmeh, S., et al. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. J. Vis. Exp. (2015)
This video demonstrates an ...