The work of Eline J. Arends and Y.K. Onno Teng is supported by the Dutch Kidney Foundation (17OKG04), Clinical Fellowship from the Netherlands Organization for Scientific Research (90713460). Laura S. van Dam&#39;s work is supported by the Foundation for Research in Rheumatology (FOREUM).

All patients and healthy controls consented to participate in the LUMC biobank. Both biobanking studies were approved by the LUMC ethical committee.
1. Isolation of Healthy Neutrophils
<ol>
	<li>Obtain 20 mL of peripheral blood from a healthy donor in two 10 mL EDTA-coated tubes.</li>
	<li>Put 10 mL of blood in a sterile 50 mL tube and add phosphate buffered saline (PBS) up to 32.5 mL.</li>
	<li>Add density gradient (e.g., Ficoll-amidotrizoaat) under the cells.
	<ol>
		<li>Take up 14 mL of density gradient with a 10 mL pipet and pipet controller.</li>
		<li>Place the pipet on the bottom of the 50 mL tube.</li>
		<li>Take the pipet controller off the pipet, allowing the density gradient to flow out of the pipet by gravity until the maximum is reached by capillary effect (1-2 mL will be left), without using the motor.</li>
		<li>Remove pipet by placing a thumb on top of the pipet, thereby preventing density gradient from leaking out during removal of the pipet.</li>
	</ol>
	</li>
	<li>Spin the tubes for 20 min at 912 x g and room temperature (RT) without acceleration or brake. 
	NOTE: Red blood cells (RBC) and neutrophils have a high density and are at the bottom of the 50 mL tube. Peripheral blood mononuclear cells (PBMCs) are separated and on top of the density gradient as a white ring. PBS-diluted plasma will be above the PBMCs. If needed, PBMCs can be isolated by transferring the white ring to a new 50 mL tube with additional washing steps with PBS.</li>
	<li>Carefully remove the white ring containing PBMCs first, followed by removal of the PBS-diluted plasma and lastly the density gradient layer as much as possible.</li>
	<li>To isolate neutrophils from the neutrophil/erythrocytes mix, lyse erythrocytes by cold sterile distilled water.
	<ol>
		<li>Take cold sterile distilled water bottle and a 10x concentrated PBS flask from the fridge.</li>
		<li>Work quickly for this step. Add 36 mL of cold sterile distilled water directly on top of the pellet and mix once carefully. Add 4 mL of 10x PBS after 20 s to make an isotonic solution. Mix once carefully.</li>
		<li>Spin tube for 5 min at 739 x g and 4 &#176;C (for the removal of RBCs). Neutrophils will be in the white pellet.</li>
		<li>Carefully discard the supernatant. Perform step 1.6.2 again and make sure the pellet is suspended properly.</li>
		<li>Spin tube for 5 min at 328 x g and 4 &#176;C.</li>
		<li>Carefully remove the supernatant and resuspend the pellet in 5 mL of PBS. Count the neutrophils and keep them on ice. 
		NOTE: Expected yield from 1 tube of blood (10 mL) is approximately 15-75 million neutrophils.</li>
	</ol>
	</li>
</ol>
2. Red Fluorescent Cell Labelling of Neutrophils
<ol>
	<li>Make a neutrophil suspension of 10-20 million neutrophils in 2 mL of PBS in a 15 mL tube.</li>
	<li>Make a solution of 2 mL PBS with 4 &#181;L of 2 &#181;M red fluorescent cell linker (see Table of Materials) in a different 15 mL tube. Add this gently to the neutrophil suspension and mix carefully.</li>
	<li>Incubate in the dark for exactly 25 min at 37 &#176;C to label the neutrophils with the red fluorescent cell linker.</li>
	<li>Inactivate the labeling by adding RPMI 1640 medium containing 10% heat inactivated fetal bovine serum (FCS)&#160;at RT up to 15 mL and mix once carefully. Make sure that the pellet is carefully resuspended if a pellet has formed.</li>
	<li>Spin tube for 5 min at 328 x g and RT.</li>
	<li>Remove the supernatant and resuspend pellet in 5 mL of phenol red-free RPMI 1640 medium containing 2% FCS and 10% P/S at RT. Count the neutrophils. 
	NOTE: A cell loss of 50% can occur after red fluorescent cell labelling.</li>
</ol>
3. Induction of Neutrophil Extracellular Trap Formation
<ol>
	<li>Make a cell suspension of 0.42 x 106 cells/mL in phenol red free RPMI 1640 medium containing 2% FCS and 10% P/S.</li>
	<li>Add 37,500 neutrophils in 90 &#181;L per well in a black 96-well, flat bottom plate.</li>
	<li>Add 10 &#181;L of the chosen stimulus (e.g., sera of patient) in triplicate to reach a concentration of 10% in the well. Always include a negative control (medium) in triplicate.</li>
	<li>Incubate in the dark at 37 &#176;C for the desired time, ranging from 30 min to 2, 4 or 6 h. Incubating for 4 h is suggested.</li>
	<li>Calculate the volume needed to add 25 &#181;L of 5 &#181;M impermeable DNA dye (see Table of Materials), to reach a final concentration of 1 &#181;M in the well. Make a predilution if necessary in RPMI 1640 medium containing 2% FCS and 10% P/S to obtain a 5 &#181;M concentration.</li>
	<li>Add 25 &#181;L of 5 &#181;M impermeable DNA dye 15 min before the end of the incubation time. Continue incubation for another 15 min at 37 &#176;C in the dark.</li>
	<li>Remove the supernatant (~125 &#181;L) very carefully and store if needed. Add 100 &#181;L of 4% paraformaldehyde (PFA). Keep in the dark, and immediately continue with step 4.</li>
</ol>
4. NET Visualization with 3D High-content, High-resolution Immunofluorescence Confocal Microscopy
<ol>
	<li>Configure the settings on the immunofluorescence confocal microscope by clicking on the Acquisition setup.
	<ol>
		<li>Click on the Configure tab.</li>
		<li>Select the objective and the camera. Choose the 10X Apo Lambda objective with an acquisition mode of a confocal 60 &#181;m pinhole.</li>
		<li>Click on Plate and choose the 96-well plastic plate. Select the sites to visit and choose a fixed number of sites. Fill in 3 columns and 3 rows without overlap (0 &#181;m), which cover a total of 45% of the well.</li>
		<li>Click on Acquisition. Select Enable Laser-Based Focusing. Select Acquire Z Series/Time Series if needed.</li>
		<li>Click on Site Autofocus.
		<ol>
			<li>Click on Focus on Plate Bottom | Off-Set by Bottom Thickness. For the initial well to find the sample, choose the first well acquired. For the site autofocus, choose all sites.</li>
		</ol>
		</li>
		<li>Click on Wavelengths.
		<ol>
			<li>For the number of wavelengths, choose 2. For the TL shading correction refinement level, choose 2.</li>
			<li>For Wavelength 1, select Texas Red.
			<ol>
				<li>For the autofocus options, select laser with Z offset, post laser offset 1.1 &#181;m. Use Z-stack with a custom range of 200-10.</li>
			</ol>
			</li>
			<li>For Wavelength 2, select FITC.
			<ol>
				<li>For the autofocus options, select laser with Z offset from w1 0 &#181;m. Use Z-stack with a custom range of 200-10.</li>
				<li>For the acquisition options, select Z series and 2D projection image maximum. For the acquisition options, select Shading Correction | Off.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Select Z Series. Select the number of steps: 10. Select the step size: 3 &#956;m (total range will be 27 &#181;m).</li>
	</ol>
	</li>
	<li>Put the plate in the immunofluorescence confocal microscopy.
	<ol>
		<li>Click on the Run tab.
		<ol>
			<li>Fill out plate name and description and choose the storage location.</li>
			<li>Select the wells that need to be acquired.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Choose exposure time for Texas Red and FITC.</li>
	<li>Click on Acquire Plate to start the acquisition, which will take approximately 1 h per plate.</li>
</ol>
5. Analysis of NET Formation
<ol>
	<li>Use an image processing program designed for analysis of scientific multidimensional images (see Table of Materials) to analyze NET formation.</li>
	<li>Transfer the acquired image data to a separate hard drive.</li>
	<li>Select the color adding tool.
	<ol>
		<li>Select w1 and choose the folder in the hard drive where the data is stored.</li>
		<li>Select w2 and choose the folder in the hard drive where the data is stored. 
		NOTE: Use a standard macro that uses w1 in the file name to add red color to the Texas Red images and uses w2 to add green color to the FITC images.</li>
	</ol>
	</li>
	<li>Select Analysis Macro.
	<ol>
		<li>Select w1, choose the threshold value (intensity threshold), which is usually 10. Select the desired pixel value (size threshold, e.g., 100).</li>
		<li>Select w2, choose the threshold value (intensity threshold), which is usually 10. Select the desired pixel value (size threshold, e.g., 500).</li>
		<li>Choose the destination for the spreadsheet file, run analysis, and save log files afterwards.</li>
	</ol>
	</li>
	<li>Analyze data in a spreadsheet.</li>
</ol>

<ol>
	<li>Jorgensen, I., Rayamajhi, M., Miao, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+as+a+defence+against+infection.">Programmed cell death as a defence against infection.</a> Nature Reviews Immunology. 17 (3), 151-164 (2017).</li><li>Rao, A. N., Kazzaz, N. M., Knight, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+neutrophil+extracellular+traps+contribute+to+the+heightened+risk+of+thrombosis+in+inflammatory+diseases?.">Do neutrophil extracellular traps contribute to the heightened risk of thrombosis in inflammatory diseases?.</a> World Journal of Cardiology. 7 (12), 829-842 (2015).</li><li>Kessenbrock, K., 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+in+autoimmune+small-vessel+vasculitis.">Netting neutrophils in autoimmune small-vessel vasculitis.</a> Nature Medicine. 15 (6), 623-625 (2009).</li><li>Garcia-Romo, G. 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=Netting+neutrophils+are+major+inducers+of+type+I+IFN+production+in+pediatric+systemic+lupus+erythematosus.">Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus.</a> Science Translational Medicine. 3 (73), 73ra20 (2011).</li><li>Lande, 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=Neutrophils+activate+plasmacytoid+dendritic+cells+by+releasing+self-DNA-peptide+complexes+in+systemic+lupus+erythematosus.">Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus.</a> Science Translational Medicine. 3 (73), 73ra19 (2011).</li><li>Meng, H., Yalavarthi, 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=In+Vivo+Role+of+Neutrophil+Extracellular+Traps+in+Antiphospholipid+Antibody-Mediated+Venous+Thrombosis.">In Vivo Role of Neutrophil Extracellular Traps in Antiphospholipid Antibody-Mediated Venous Thrombosis.</a> Arthritis &amp; Rheumatology. 69 (3), 655-667 (2017).</li><li>Desai, 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=Particles+of+different+sizes+and+shapes+induce+neutrophil+necroptosis+followed+by+the+release+of+neutrophil+extracellular+trap-like+chromatin.">Particles of different sizes and shapes induce neutrophil necroptosis followed by the release of neutrophil extracellular trap-like chromatin.</a> Scientific Reports - Nature. 7 (1), 15003 (2017).</li><li>Desai, 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=PMA+and+crystal-induced+neutrophil+extracellular+trap+formation+involves+RIPK1-RIPK3-MLKL+signaling.">PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling.</a> European Journal of Immunology. 46 (1), 223-229 (2016).</li><li>Apel, F., Zychlinsky, A., Kenny, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+neutrophil+extracellular+traps+in+rheumatic+diseases.">The role of neutrophil extracellular traps in rheumatic diseases.</a> Nature Reviews Rheumatology. , (2018).</li><li>Kraaij, 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=A+novel+method+for+high-throughput+detection+and+quantification+of+neutrophil+extracellular+traps+reveals+ROS-independent+NET+release+with+immune+complexes.">A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.</a> Autoimmunity Reviews. 15 (6), 577-584 (2016).</li><li>Masuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NETosis+markers:+Quest+for+specific,+objective,+and+quantitative+markers.">NETosis markers: Quest for specific, objective, and quantitative markers.</a> Clinica Chimica Acta. 459, 89-93 (2016).</li><li>Brinkmann, V., Goosmann, C., Kuhn, L. I., 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=Automatic+quantification+of+in+vitro+NET+formation.">Automatic quantification of in vitro NET formation.</a> Frontiers in Immunology. 3, 413 (2012).</li><li>Nakazawa, D., 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=Enhanced+formation+and+disordered+regulation+of+NETs+in+myeloperoxidase-ANCA-associated+microscopic+polyangiitis.">Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCA-associated microscopic polyangiitis.</a> Journals of the American Society of Nephrology. 25 (5), 990-997 (2014).</li><li>Konig, M. F., Andrade, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Reappraisal+of+Neutrophil+Extracellular+Traps+and+NETosis+Mimics+Based+on+Differential+Requirements+for+Protein+Citrullination.">A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination.</a> Frontiers in Immunology. 7, 461 (2016).</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, (2017).</li><li>Zhao, W., Fogg, D. K., Kaplan, M. J. <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+image-based+quantitative+method+for+the+characterization+of+NETosis.">A novel image-based quantitative method for the characterization of NETosis.</a> Journal of Immunological Methods. 423, 104-110 (2015).</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> The Journal of Immunology. 185 (12), 7413-7425 (2010).</li><li>Tanaka, K., 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+characterization+of+neutrophil+extracellular+traps+in+various+organs+of+a+murine+sepsis+model.">In vivo characterization of neutrophil extracellular traps in various organs of a murine sepsis model.</a> PLoS One. 9 (11), e111888 (2014).</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> Journal of Cell Biology. 176 (2), 231-241 (2007).</li><li>von K&#246;ckritz-Blickwede, M., Chow, O., Nizet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+calf+serum+contains+heat-stable+nucleases+that+degrade+neutrophil+extracellular+traps.">Fetal calf serum contains heat-stable nucleases that degrade neutrophil extracellular traps.</a> Blood. 114 (25), 5245-5246 (2009).</li><li>Kraaij, 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=Excessive+neutrophil+extracellular+trap+formation+in+ANCA-associated+vasculitis+is+independent+of+ANCA.">Excessive neutrophil extracellular trap formation in ANCA-associated vasculitis is independent of ANCA.</a> Kidney International. , (2018).</li><li>Kraaij, 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=The+NET-effect+of+combining+rituximab+with+belimumab+in+severe+systemic+lupus+erythematosus.">The NET-effect of combining rituximab with belimumab in severe systemic lupus erythematosus.</a> Journal of Autoimmunity. , (2018).</li><li>Hahn, J., Schauer, 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=Aggregated+neutrophil+extracellular+traps+resolve+inflammation+by+proteolysis+of+cytokines+and+chemokines+and+protection+from+antiproteases.">Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.</a> The FASEB Journal. (Aug 21), (2018).</li></ol>

The authors have nothing to disclose.

The most critical part of this assay is the need to use freshly isolated neutrophils for each experiment because neutrophils are short-lived and die when frozen. This requires a healthy donor each time, which could implicate some variations due to donor characteristics. One of these variations is the activation status of the neutrophils. Neutrophils could be activated already in vivo prior to isolation. In addition, neutrophils can be activated throughout the isolation steps notably during lysis of erythrocytes, therefore an experienced handler of neutrophils is required to minimize the activation of neutrophils. In general, isolation of neutrophils should be performed as soon as possible after blood drawing and the experiment should not be paused to avoid excessive spontaneous activation. Secondly, rough handling of neutrophils should be avoided. As such, the notable advantage of the described protocol is the minimal pipetting interventions once neutrophils are seeded in a 96-well plate. Importantly, the activation status of the neutrophils is best assessed in the unstimulated condition, in which this assay can sensitively detect low levels of NET formation. Another factor that could influence the assay is the use of FCS in the medium. The percentage of FCS has been decreased from 10% to 2% to avoid the possible suppression of NET formation by antioxidant activity19,20 or the possible activation of the neutrophils despite heat inactivation. Culture without FCS or with the use different types of media has not been tried. An unstimulated or medium control is always taken along when performing the assay to have an indication of the background signal (e.g., activation status of the neutrophils). The fold increase for each stimulus compared to the unstimulated sample is displayed to achieve consistent results over different experiments using the same stimulus.
An important factor for a possible high background is staining of extracellular DNA that is unrelated to the process of NET formation. The present assay attempts to reduce this by removing the extracellular DNA staining immediately after the short incubation period of 15 min and by analyzing the plate directly after fixation. Therefore, it is essential to use an advanced confocal microscope that has enough speed and analytical power to capture the 96-well plate within 1 to 2 h. Automated setting of the exposure time and focus is recommended. As such, the microscope setting can vary between each sample and experiment with respect to the color intensity threshold which is necessary for overall optimal picture quality. The latter influences the eventual capability to correctly quantify neutrophils and NETs and the optimal setting should therefore be confirmed by using multiple control samples (e.g.,&#160;serum of healthy controls). During the analysis of captured images, the use of a pixel threshold and size threshold in the analysis program allows for a better selection of NET formation.
Extracellular DNA derived from NET formation can be the result of distinct death pathways, including NETosis, necroptosis, pyroptosis, ferroptosis or even non-lytic process of vital NET formation1. As such, a limitation of the present assay is that by staining only for extracellular DNA there is no differentiation possible between NET formation and other relevant cell death pathways. It is possible to achieve this by using either selective inhibitors of distinct death pathways to discriminate between the different forms underpinning NET formation or confirm by separate immunostainings the presence of specific NET markers, such as citH3 and NE, co-localized with DNA. The co-localization of extracellular DNA with citH3 and NE has recently been confirmed for this assay10. The advantage of avoiding NET specific markers in this assay, allows to assess all forms of NET formation leading to the extrusion of DNA by neutrophils as complete and as objective as possible with the potential of high-throughput screening. The applicability of this protocol has been shown in studying low level NET induction mediated by immune complexes in auto-immune disease in which the ability to detect qualitative and quantitative differences might be more important than the type of process involved10,21,22. Illustrating that this novel NET quantification assay can be of added value for different researchers to investigate various aspects of NET formation. Small adjustments to the assay are easily implemented: adjustment of the stimulation period, the use of a favorite NET marker to focus on one specific death pathway leading to NET formation, the use of a different magnification or the use of different NET criteria in the quantification and analysis.
In conclusion, the protocol provided is a highly-sensitive broadly applicable assay for the semi-automated quantification of NET formation for the evaluation of ex vivo induction of NETs upon different stimuli.

The formation of neutrophil extracellular traps (NETs) is the process in which neutrophils release their DNA in an extracellular three-dimensional (3D) web like structure, complexed with a wide range of antimicrobial and dangerous molecules, granular and cytoplasmic enzymes, peptides and proteins. These immunogenic and toxic structures have an important physiological role in the innate immune defense of healthy individuals by trapping and killing infectious pathogens1. However, they have also been demonstrated to be involved in thrombosis2 and various systemic autoimmune diseases, including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV)3, systemic lupus erythematosus (SLE)4,5, antiphospholipid syndrome (APS)2,6, rheumatoid arthritis (RA), psoriasis, and gout7,8,9.
In vitro NET formation has been widely studied with the chemical compound phorbol 12-myristate 13-acetate (PMA), which induces massive NET formation. However most physiological stimuli induce much lower levels of NET formation10. To study NET-triggers in, for example, an autoimmune disease setting, a standardized, sensitive, high-throughput quantitative assay is needed to detect and quantify NET formation. Quantification of NETs has proven to be challenging and is currently performed by different methods, each with their own advantages and limitations11. A commonly used method is the detection of DNA in the supernatants12, which is objective but does not discriminate between the origin of the DNA (apoptotic, necrotic, NETs), and therefore is not very specific for NETs. Secondly, enzyme-linked immunosorbent assays (ELISAs) of DNA-complexed with NET-specific proteins, for example, myeloperoxidase (MPO) or neutrophil elastase (NE), are a more specific approach to detect NETs and were demonstrated to correlate well with citrullinated histone-3 (CitH3) positive NETs13. However, it is not known whether this method is sensitive enough to pick up all NETs (e.g.,&#160;MPO, NE, and CitH3 negative NETs). A third approach is immunofluorescence microscopy that is used to detect co-localization of NET-associated molecules (NE, MPO, CitH3) with extracellular DNA to quantify NETs. This method is generally specific for NETs, but it cannot be applied as a high-throughput method and is not objective due to observer bias. Additionally, this method does not take into account MPO-, NE-, CitH3-negative NETs that are frequently present depending on the used NET-trigger14,15. Flow cytometry approaches detect NETs through a changed forward/sideward scatter (FSC/SSC) indicating swelling of the nucleus in NET-ting neutrophils16. This method does not take into account the different forms of NET formation that have been identified, which might not involve swelling of the nucleus, such as vital NET formation17. Lastly, immunofluorescence confocal microscopy has been applied to visualize and quantify NET formation by directly staining extracellular DNA with a cell-impermeable dye that stains extracellular DNA12,18. Generally, 5 to 10 high-power fields are manually picked and assessed, which covers 1-5% of each well of a 96-well plate11,17. Manual selection of images is not always objective, prone to bias and not attractive for high-throughput analysis. An automated, high-throughput NET quantification assay was recently developed, which imaged 11% of the well in a 3D manner covering 13 &#181;m through Z-stacked immunofluorescence confocal microscopy, thereby leading to a highly sensitive technique to assess NETs compared to the conventional methods10. The current report describes the most recent protocol to quantify NET formation through an automated, highly sensitive assay using 3D confocal microscopy, which achieves a total imaged area of 45% of each well and covers 27 &#181;m through Z-stacks. This protocol is suitable to quantify, with a high sensitivity, low levels of NET formation in an objective and unbiased manner.

<table>
	<tbody>
		<tr>
			<td>Aqua Sterile H2O</td>
			<td>B. Braun, Melsungen, Germany</td>
			<td>12604052</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FCS)</td>
			<td>Bodinco, Alkmaar, The Netherlands</td>
			<td></td>
			<td>Used in high concentrations it could influcence NET formation</td>
		</tr>
		<tr>
			<td>Ficoll 5,7% - amidotrizoaat 9% density 1,077 g / mL</td>
			<td>LUMC, Leiden, The Netherlands</td>
			<td>97902861</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunofluorescence confocal microscope</td>
			<td>Image Xpress Micro Confocal (Molecular Devices, Sunnyvale, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neutralization PBS (10x)</td>
			<td>Gibco, Paisley, UK</td>
			<td>70011-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / streptomycin (p/s)</td>
			<td>Gibco, Paisley, UK</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red free RPMI 1640 (1x)</td>
			<td>Gibco, Paisley, UK</td>
			<td>11835-063</td>
			<td>Phenol red can interfere with the immunofluorescence signal</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>B. Braun, Melsungen, Germany</td>
			<td>174628062</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 2 uM Red fluorescent cell linker</td>
			<td>Sigma Aldrich Saint-Louis, MO, USA</td>
			<td>PKH26GL-1KT</td>
			<td>PKH are patented fluorescent dyes and a cell labeling technology named after their discoverer Paul Karl Horan</td>
		</tr>
		<tr>
			<td>Program for scientific multidimensional images analysis</td>
			<td>ImageJ, Research Services Branch, National Institute of Mental Health, Bethesda, Maryland, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 (1x)</td>
			<td>Gibco, Paisley, UK</td>
			<td>52400-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Sytox green 5 mM Impermeable DNA dye</td>
			<td>Gibco, Paisley, UK</td>
			<td>57020</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain 0,4%</td>
			<td>Sigma Aldrich, Germany</td>
			<td>17942E</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well, black, flat bottom, tissue culture treated plate</td>
			<td>Falcon, NY, USA</td>
			<td>353219</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Neutrophil extracellular trap (NET) formation is quantified in a 3D manner by quantifying stained extracellular DNA over 10 Z-stacks with 3 &#181;m distance starting off at the focal plane in each well. By measuring the cumulative area, the sensitivity of the assay increases (Figure 1A). The isolated neutrophils have a mean purity of 98.7% with standard deviation (SD) of 1.10% measured in 14 different samples from different isolations. The mean percentage of red blood cells is 1.04% &#177; 1.1% SD and the mean percentage of monocytes is 0.085% &#177; 0.17% SD (data not shown). The total area of stained neutrophils imaged are quantified only in the focal plane in each well, which correlate significantly with the total neutrophil count in each well with a Pearson correlation coefficient of 0.99 (95% confidence interval [CI] 0.985-0.997, p&#160;&#60; 0.0001) (Figure 1B). The representative outcome of quantification of NET formation in neutrophils stimulated with 10% sera of AAV patients or medium (MED) as a negative control, expressed as cumulative stained extracellular DNA area over 10 Z-stacks per imaged neutrophil (Figure 1C). Snapshots of representative images of unstimulated neutrophils (Figure 2A) and of NETs in AAV-stimulated neutrophils (Figure 2B) are shown.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59150/59150fig1.jpg" /> 
Figure 1: Quantification of NET formation by measuring extracellular DNA and neutrophil count. (A)&#160;Area is cumulatively quantified over the 10 Z-stacks for each well for unstimulated neutrophils (MED) and for neutrophils stimulated with 10% serum of ANCA-associated vasculitis (AAV) patients (n = 4) quantified with an image processing program. Each stimulus was tested in triplicate, each point represents the median value. (B) Red fluorescent labelled cell area and cell count were quantified in the focal plane of each well by the image processing program (R2 = 0.99, p&#160;&#60; 0.0001). (C)&#160;NET formation is expressed as cumulative area per imaged neutrophil (cell area). The mean &#177; standard error of the mean (SEM) of each triplicate is plotted per stimulus. <a href="https://www.jove.com/files/ftp_upload/59150/59150fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59150/59150fig2.jpg" /> 
Figure 2: Snapshots of NET quantification assay. Fluorescent labelled neutrophils are shown in red, and stained extracellular DNA is shown in green. 10x Plan Apo Lambda objective. (A) Unstimulated neutrophils. (B) Neutrophils stimulated with AAV serum. <a href="https://www.jove.com/files/ftp_upload/59150/59150fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation at JoVE.com

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

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. ...

a-high-throughput-assay-to-assess-quantify-neutrophil-extracellular

Research

JoVE Journal

Immunology and Infection

9.9K Views.  Leiden University Medical Center. 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.

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

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 ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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

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

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

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

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

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

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

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

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

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

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

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

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

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

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

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...