Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Summary

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.

Abstract

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.

Introduction

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

Protocol

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 a sufficiently large cell pellet.
2. Mix the blood with one volume of 2% dextran in saline (0.9% NaCl) and allow to sediment at room temperature....

Results

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.......

Discussion

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 areas^5,7,14,15. Acquisition of images using Immunofluorescence microscopy and subsequent image-based quantification is a widely used method to quantify NETs. This approach has.......

Materials

List of materials used in this article

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