JoVE Logo
Faculty Resource Center

Sign In





Representative Results





Immunology and Infection

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Published: November 27th, 2019



1Department of Clinical Sciences, Division of Infection Medicine, Lund University

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. 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.

Neutrophils are crucial mediators of innate host defense responses against a wide variety of microbial pathogens1. They execute their antimicrobial functions by releasing their granules containing a wide array of antimicrobial proteins2, producing reactive oxygen species (ROS) and hypochlorite1, and through phagocytosis3. In addition, Brinkmann et al.4 described neutrophil extracellular traps (NETs) as a novel mechanism by which neutrophils trap and eliminate invading pathogens. Since their discovery a little over a decade ago.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The ethics committee of Lund University approved the collection of venous blood from healthy volunteers in accordance with the Declaration of Helsinki (2013/728). All volunteers provided their written informed consent.

1. Isolation of Peripheral Blood Neutrophils using Density-Gradient Centrifugation

  1. Collect human venous blood in tubes containing heparin and allow the tubes to reach room temperature.
    Note: A minimum of 16 mL of blood from a healthy donor is required to yield.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

5 x 105 neutrophils/mL were seeded onto coverslips placed in a 12-well plate and stimulated with either 20 nM PMA or left unstimulated for 150 min. The samples were then stained using primary rabbit anti-human neutrophil elastase antibodies, secondary goat anti-rabbit fluorophore conjugated antibodies and DAPI - a fluorescently labelled dye that stains DNA (See the Table of Materials for details). A minimum of 5 images were then acquired using an epifluorescenc.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NET formation is a relatively recent addition to the diverse neutrophil armamentarium4 and there has been a noticeable surge of interest to study the implication of NETs in a wide array of research areas5,7,14,15. Acquisition of images using Immunofluorescence microscopy and subsequent image-based quantification is a widely used method to quantify NETs. This approach has.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The work was funded by the Crafoord Foundation (TM and PN), Swedish Government Research grant (PN, TM), Swedish research council (PN) and Groschinsky Foundation (TM, PN).


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
BD Vacutainer Heparinised plastic tubes BD Biosciences 367885
Lymphoprep Axis-Shield 114547
RPMI-1640 with L-Glutamine Gibco 11835-030
50mL conical flasks Sarstedt 62.547.004
15mL conical flasks Sarstedt 62.554.002
12-well Tissue culture plates Falcon 10626491
#1 Coverslips 10mm Menzel Glaser CS10100
Glass slides Menzel Glaser 631-0098
Primary anti-human elastase DAKO DAKO rabbit 1373, contract immunization
Secondary fluorophore conjugated goat anti-rabbit Life technologies A-11072, A-11070
PROLONG-Gold Antifade reagent with DAPI Life technologies P36930 Mounting medium
Goat serum Sigma-Aldrich G9023
Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich 79346
Paraformaldehyde Sigma-Aldrich 158127
Triton X-100 Sigma-Aldrich T8787
Nikon Ti-E Epifluorescence microscope Nikon
CCD camera Andor Zyla
Plan Apochromat 20x, 40x objectives Nikon
Windows 10 Microsoft Operating system
macOS Sierra 10.12 Apple Operating system
MATLAB Mathworks

  1. Nauseef, W. M., Borregaard, N. Neutrophils at work. Nature Immunology. 15 (7), 602-611 (2014).
  2. Borregaard, N., Cowland, J. B. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood. 89 (10), 3503-3521 (1997).
  3. Nordenfelt, P., Tapper, H. Phagosome dynamics during phagocytosis by neutrophils. Journal of Leukocyte Biology. 90 (2), 271-284 (2011).
  4. Brinkmann, V., et al. Neutrophil extracellular traps kill bacteria. Science. 303 (5663), 1532-1535 (2004).
  5. Sorensen, O. E., Borregaard, N. Neutrophil extracellular traps - the dark side of neutrophils. Journal of Clinical Investigation. 126 (5), 1612-1620 (2016).
  6. Yipp, B. G., Kubes, P. NETosis: how vital is it?. Blood. 122 (16), 2784-2794 (2013).
  7. Jorch, S. K., Kubes, P. An emerging role for neutrophil extracellular traps in noninfectious disease. Nature Medicine. 23 (3), 279-287 (2017).
  8. Fuchs, T. A., et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol. 176 (2), 231-241 (2007).
  9. Mohanty, T., et al. A novel mechanism for NETosis provides antimicrobial defense at the oral mucosa. Blood. 126 (18), 2128-2137 (2015).
  10. Hakkim, A., et al. Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation. Nature Chemical Biology. 7 (2), 75-77 (2011).
  11. Brinkmann, V., Goosmann, C., Kuhn, L. I., Zychlinsky, A. Automatic quantification of in vitro NET formation. Frontiers in Immunology. 3, 413 (2012).
  12. Coelho, L. P., et al. Automatic determination of NET (neutrophil extracellular traps) coverage in fluorescent microscopy images. Bioinformatics. 31 (14), 2364-2370 (2015).
  13. Mohanty, T., Sorensen, O. E., Nordenfelt, P. NETQUANT: Automated Quantification of Neutrophil Extracellular Traps. Frontiers in Immunology. 8, 1999 (2017).
  14. Kaplan, M. J., Radic, M. Neutrophil extracellular traps: double-edged swords of innate immunity. Journal of Immunology. 189 (6), 2689-2695 (2012).
  15. Cedervall, J., Olsson, A. K. Immunity Gone Astray - NETs in Cancer. Trends in Cancer. 2 (11), 633-634 (2016).
  16. Ginley, B. G., et al. Computational detection and quantification of human and mouse neutrophil extracellular traps in flow cytometry and confocal microscopy. Scientific Reports. 7 (1), 17755 (2017).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved