DNA Area and NETosis Analysis (DANA): a High-Throughput Method to Quantify Neutrophil Extracellular Traps in Fluorescent Microscope Images

Abstract

BackgroundNeutrophil extracellular traps (NETs), extracellular structures composed of decondensed chromatin and antimicrobial molecules, are released in a process called NETosis. NETs, which are part of normal host defense, have also been implicated in multiple human diseases. Unfortunately, methods for quantifying NETs have limitations which constrain the study of NETs in disease. Establishing optimal methods for NET quantification holds the potential to further elucidate the role of NETs in normal and pathologic processes.ResultsTo better quantify NETs and NET-like structures, we created DNA Area and NETosis Analysis (DANA), a novel ImageJ/Java based program which provides a simple, semi-automated approach to quantify NET-like structures and DNA area. DANA can analyze many fluorescent microscope images at once and provides data on a per cell, per image, and per sample basis. Using fluorescent microscope images of Sytox-stained human neutrophils, DANA quantified a similar frequency of NET-like structures to the frequency determined by two different individuals counting by eye, and in a fraction of the time. As expected, DANA also detected increased DNA area and frequency of NET-like structures in neutrophils from subjects with rheumatoid arthritis as compared to control subjects. Using images of DAPI-stained murine neutrophils, DANA (installed by an individual with no programming background) gave similar frequencies of NET-like structures as the frequency of NETs determined by two individuals counting by eye. Further, DANA quantified more NETs in stimulated murine neutrophils compared to unstimulated, as expected.ConclusionsDANA provides a means to quantify DNA decondensation and the frequency of NET-like structures using a variety of different fluorescent markers in a rapid, reliable, simple, high-throughput, and cost-effective manner making it optimal to assess NETosis in a variety of conditions.Electronic supplementary materialThe online version of this article (10.1186/s12575-018-0072-y) contains supplementary material, which is available to authorized users.

IL-4 receptor engagement in human neutrophils impairs their migration and extracellular trap formation

“…For automatic analysis, we used ImageJ software with the DANA plug-in to quantify the area, raw integrated density, aspect ratio, roundness, maximum and minimum brightness, and solidity of each region of interest of 49,6-diamidino-2phenylindole dihydrochloride-labeled neutrophils, as previously described. 30…”

Convolutional Neural Networks–Based Image Analysis for the Detection and Quantification of Neutrophil Extracellular Traps

“…Furthermore, there are multiple methodologies used to quantitatively assess the process of NETs formation-some of them relying on the measurement of the extracellular DNA using DNA-intercalating dyes, some of them using ELISA measurements of DNA complexed with neutrophil-derived proteins, and others are based on the analysis of microscopical images with manual or automated counting [10,[54][55][56]. Even though a number of automated or semi-automated methodologies to quantify NETs based on microscopy images have been proposed [11,14,20], some of them relatively easy to implement on a large scale [12,13,17], still very few of them have been widely adopted by other researchers. Recently, an imaging flow cytometry has been proposed as a method for automatic determination of NETosis [21,57].…”

“…What is more, simple immune processing of the samples would not allow to differentiate, whether changes in the nucleus morphology coincide with the disruption of plasma membrane. Live imaging of cells appears to be a good alternative to immune labelling, since it requires less processing and entails much lower risk of introducing artifacts [13,17,19]. However, immune-histological staining may be necessary to confirm the presence of NETs if uncommon inducers are used or in samples consisting of mixed cell populations (not solely isolated granulocytes).…”

“…However, immune-histological staining may be necessary to confirm the presence of NETs if uncommon inducers are used or in samples consisting of mixed cell populations (not solely isolated granulocytes). One should also bear in mind that NETs composition may differ and not all of the markers commonly used to identify NETs might be detected on all immunofluorescently-labeled NET-like structures [13].…”

“…Accordingly, several authors provided digital, semi-or fully automatic solutions to facilitate the quantitative analysis of microscopic images. Most of the methodologies described previously rely on an analysis of paraformaldehyde-fixed samples stained either solely with DNA-binding dyes or in combination with immunofluorescently labeled antibodies [11][12][13][14][15][16]. However, one should bear in mind that fixation procedure precludes differentiation between dead and viable cells [17].…”