{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T13:46:46Z","timestamp":1787060806607,"version":"build-2736575974"},"reference-count":40,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T00:00:00Z","timestamp":1773187200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>NETosis is a distinct form of neutrophil cell death involved in innate immunity and characterized by the release of DNA, that when dysregulated contributes to tissue damage and target-organ injury. As our understanding of the mechanisms governing this process continues to advance, there is a growing need for refined tools that can precisely characterize NETosis and enable efficient screening of its modulators. Here, we present a novel live-cell imaging and analysis pipeline for quantifying NETosis in cultured cells. We have generated a CellProfiler pipeline that enables in-depth analysis of cell and NET features, allowing for subsequent characterization and classification of NETosis stages using machine learning. Coupled with high throughput live cell imaging, this approach allows for large-scale and automated tracking of NETosis progression. The pipeline was validated in promyelocytic HL-60 cells differentiated into granulocyte-like cells, as well as primary neutrophils from the bone marrow of mice. We further confirmed dose and stimulus-dependent responses to common stimuli and pharmacological inhibitors of NETosis. As a flexible and scalable high-throughput imaging pipeline, our novel approach allows for the assessment of NETting dynamics and the screening of potential NETosis-modulating agents, which will be instrumental in developing therapies for NET-induced tissue injury.<\/jats:p>","DOI":"10.3389\/fimmu.2026.1753477","type":"journal-article","created":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T05:36:14Z","timestamp":1773207374000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["A semi-automated imaging and analysis pipeline for NET quantification and temporal-profiling of NETosis"],"prefix":"10.3389","volume":"17","author":[{"given":"Chlo\u00e9","family":"Landry","sequence":"first","affiliation":[{"name":"Kidney Research Centre, Inflammation and Chronic Disease Program, The Ottawa Hospital Research Institute","place":["Ottawa, ON, Canada"]},{"name":"Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa","place":["Ottawa, ON, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liyuan","family":"Wang","sequence":"additional","affiliation":[{"name":"Cell Biology and Image Acquisition Core Facility, Faculty of Medicine, University of Ottawa","place":["Ottawa, ON, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emma","family":"Gerber","sequence":"additional","affiliation":[{"name":"Kidney Research Centre, Inflammation and Chronic Disease Program, The Ottawa Hospital Research Institute","place":["Ottawa, ON, Canada"]},{"name":"Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa","place":["Ottawa, ON, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chet Elliot","family":"Holterman","sequence":"additional","affiliation":[{"name":"Kidney Research Centre, Inflammation and Chronic Disease Program, The Ottawa Hospital Research Institute","place":["Ottawa, ON, Canada"]},{"name":"Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa","place":["Ottawa, ON, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dylan","family":"Burger","sequence":"additional","affiliation":[{"name":"Kidney Research Centre, Inflammation and Chronic Disease Program, The Ottawa Hospital Research Institute","place":["Ottawa, ON, Canada"]},{"name":"Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa","place":["Ottawa, ON, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,3,11]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s41392-024-01933-x","article-title":"Neutrophil extracellular traps in homeostasis and disease","volume":"9","author":"Wang","year":"2024","journal-title":"Signal Transduct Target Ther"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.116.309692","article-title":"Neutrophil extracellular traps in atherosclerosis and atherothrombosis","volume":"120","author":"D\u00f6ring","year":"2017","journal-title":"Circ Res"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1093\/CVR\/CVAB329","article-title":"Neutrophil extracellular traps: from physiology to pathology","volume":"118","author":"Hidalgo","year":"2022","journal-title":"Cardiovasc Res"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1155\/2022\/9339411","article-title":"Cell-Free DNA, neutrophil extracellular traps (NETs), and endothelial injury in coronavirus disease 2019- (COVID-19-) associated acute kidney injury","volume":"2022","author":"Henry","year":"2022","journal-title":"Mediators Inflammation"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.4049\/JIMMUNOL.1201719","article-title":"Neutrophil extracellular traps: double-edged swords of innate immunity","volume":"189","author":"Kaplan","year":"2012","journal-title":"J Immunol"},{"key":"B6","doi-asserted-by":"publisher","first-page":"1724","DOI":"10.3791\/1724","article-title":"Neutrophil extracellular traps: how to generate and visualize them","volume":"36","author":"Brinkmann","year":"2010","journal-title":"J Vis Exp"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.3389\/FIMMU.2024.1459933","article-title":"NETQUANT2: automated web-based quantification of neutrophil extracellular traps from fluorescence microscopy","volume":"15","author":"Kumra Ahnlide","year":"2024","journal-title":"Front Immunol"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.3389\/FIMMU.2012.00413","article-title":"Automatic quantification of in vitro NET formation","volume":"3","author":"Brinkmann","year":"2012","journal-title":"Front Immunol"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1186\/s12575-018-0072-y","article-title":"DNA area and NETosis analysis (DANA): a high-throughput method to quantify neutrophil extracellular traps in fluorescent microscope images","volume":"20","author":"Rebernick","year":"2018","journal-title":"Biol Proced Online"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btv156","article-title":"Automatic determination of NET (neutrophil extracellular traps) coverage in fluorescent microscopy images","volume":"31","author":"Coelho","year":"2015","journal-title":"Bioinformatics"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.3389\/FIMMU.2017.01999","article-title":"NETQUANT: Automated quantification of neutrophil extracellular traps","volume":"8","author":"Mohanty","year":"2018","journal-title":"Front Immunol"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.3390\/cells9020508","article-title":"Convolutional neural networks-based image analysis for the detection and quantification of neutrophil extracellular traps","volume":"9","author":"Manda-Handzlik","year":"2020","journal-title":"Cells"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1073\/PNAS.1909546117\/SUPPL_FILE\/PNAS.1909546117.SM22.AVI","article-title":"NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture","volume":"117","author":"Thiama","year":"2020","journal-title":"Proc Natl Acad Sci USA"},{"key":"B14","doi-asserted-by":"publisher","first-page":"12355","DOI":"10.3390\/ijms241512355","article-title":"Stages of NETosis development upon stimulation of neutrophils with activators of different types","volume":"24","author":"Inozemtsev","year":"2023","journal-title":"Int J Mol Sci"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1083\/JCB.200606027","article-title":"Novel cell death program leads to neutrophil extracellular traps","volume":"176","author":"Fuchs","year":"2007","journal-title":"J Cell Biol"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.3389\/FIMMU.2023.1021638\/FULL","article-title":"Trapalyzer: a computer program for quantitative analyses in fluorescent live-imaging studies of neutrophil extracellular trap formation","volume":"14","author":"Ciach","year":"2023","journal-title":"Front Immunol"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1038\/S41592-019-0582-9","article-title":"ilastik: interactive machine learning for (bio)image analysis","volume":"16","author":"Berg","year":"2019","journal-title":"Nat Methods"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1186\/S12859-021-04344-9","article-title":"CellProfiler 4: improvements in speed, utility and usability","volume":"22","author":"Stirling","year":"2021","journal-title":"BMC Bioinf"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1093\/BIOINFORMATICS\/BTAB634","article-title":"CellProfiler Analyst 3.0: accessible data exploration and machine learning for image analysis","volume":"37","author":"Stirling","year":"2021","journal-title":"Bioinformatics"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1038\/NCHEMBIO.1735","article-title":"Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation","volume":"11","author":"Lewis","year":"2015","journal-title":"Nat Chem Biol"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1371\/JOURNAL.PONE.0022043","article-title":"PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection","volume":"6","author":"Hemmers","year":"2011","journal-title":"PloS One"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1073\/PNAS.75.5.2458","article-title":"Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds","volume":"75","author":"Collins","year":"1978","journal-title":"Proc Natl Acad Sci USA"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1126\/SCIENCE.1101400","article-title":"Human PAD4 regulates histone arginine methylation levels via demethylimination","volume":"306","author":"Wang","year":"2004","journal-title":"Science"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1084\/JEM.20100239","article-title":"PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps","volume":"207","author":"Li","year":"2010","journal-title":"J Exp Med"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.3791\/53936","article-title":"Murine hind limb long bone dissection and bone marrow isolation","volume":"2016","author":"Amend","year":"2016","journal-title":"J Vis Exp"},{"key":"B26","doi-asserted-by":"publisher","first-page":"877","DOI":"10.1002\/2211-5463.12222","article-title":"Spontaneous formation of neutrophil extracellular traps in serum\u2010free culture conditions","volume":"7","author":"Kamoshida","year":"2017","journal-title":"FEBS Open Bio"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.3389\/FIMMU.2023.1301183","article-title":"Isolation methods determine human neutrophil responses after stimulation","volume":"14","author":"Kr\u00e9mer","year":"2023","journal-title":"Front Immunol"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.3389\/FIMMU.2022.820058","article-title":"Method matters: effect of purification technology on neutrophil phenotype and function","volume":"13","author":"Blanter","year":"2022","journal-title":"Front Immunol"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.3389\/FIMMU.2021.767175\/XML","article-title":"Neutrophils\u2014From bone marrow to first-line defense of the innate immune system","volume":"12","author":"Kraus","year":"2021","journal-title":"Front Immunol"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1016\/J.SMIM.2023.101849","article-title":"Regulated cell death in neutrophils: from apoptosis to NETosis and pyroptosis","volume":"70","author":"Dejas","year":"2023","journal-title":"Semin Immunol"},{"key":"B31","doi-asserted-by":"publisher","first-page":"3767","DOI":"10.1038\/s41467-018-06263-5","article-title":"Chromatin swelling drives neutrophil extracellular trap release","volume":"9","author":"Neubert","year":"2018","journal-title":"Nat Commun"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1016\/J.HELIYON.2023.E16982","article-title":"Comparison of NET quantification methods based on immunofluorescence microscopy: hand-counting, semi-automated and automated evaluations","volume":"9","author":"Henneck","year":"2023","journal-title":"Heliyon"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1371\/JOURNAL.PONE.0176472","article-title":"In vitro induction of NETosis: comprehensive live imaging comparison and systematic review","volume":"12","author":"Hoppenbrouwers","year":"2017","journal-title":"PloS One"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.21769\/BioProtoc.2386","article-title":"In vitro demonstration and quantification of neutrophil extracellular trap formation","volume":"7","author":"Jiang","year":"2017","journal-title":"Bio Protoc"},{"key":"B35","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1561\/0600000035","article-title":"Decision forests: a unified framework for classification, regression, density estimation, manifold learning and semi-supervised learning","volume":"7","author":"Criminisi","year":"2012","journal-title":"Foundations Trends\u00ae Comput Graphics Vision"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1002\/lio2.396","article-title":"MUC5B induces in vitro neutrophil extracellular trap formation: implication in otitis media","volume":"5","author":"Val","year":"2020","journal-title":"Laryngoscope Investig Otolaryngol"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1039\/C5IB00225G","article-title":"Microfluidic device for simultaneous analysis of neutrophil extracellular traps and production of reactive oxygen species","volume":"8","author":"Moussavi-Harami","year":"2016","journal-title":"Integr Biol"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.3389\/FIMMU.2019.02313","article-title":"Compromised anti-inflammatory action of neutrophil extracellular traps in PAD4-deficient mice contributes to aggravated acute inflammation after myocardial infarction","volume":"10","author":"Eghbalzadeh","year":"2019","journal-title":"Front Immunol"},{"key":"B39","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1038\/s41418-018-0261-x","article-title":"To NET or not to NET: current opinions and state of the science regarding the formation of neutrophil extracellular traps","volume":"26","author":"Boeltz","year":"2019","journal-title":"Cell Death Differ"},{"key":"B40","doi-asserted-by":"publisher","first-page":"2211","DOI":"10.1007\/S00018-016-2195-0","article-title":"Matters of life and death. How neutrophils die or survive along NET release and is \u201cNETosis\u201d = necroptosis","volume":"73","author":"Desai","year":"2016","journal-title":"Cell Mol Life Sci"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1753477\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T05:36:16Z","timestamp":1773207376000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1753477\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,11]]},"references-count":40,"alternative-id":["10.3389\/fimmu.2026.1753477"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2026.1753477","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,11]]},"article-number":"1753477"}}