A sensitive, fast, and efficient method for studying NETosis ex vivo by flow cytometry and live cell imaging 2450 Article Swipe

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Susannah H. Kassmer , Anagha Divekar , Xifeng Yang ·

YOU? · · 2025 · Open Access · · DOI: https://doi.org/10.1093/jimmun/vkaf283.367

Description In a process termed NETosis, neutrophils expel neutrophil extracellular traps (NETs) - chromatin fibers decorated with antimicrobials and proteases, including neutrophil elastase - in an effort to trap and eliminate pathogens. NETosis plays a role in the pathogenesis of multiple diseases, and standardized methods for analyzing and measuring the process are urgently needed for the advancement of this research area. Here we demonstrate a sensitive and streamlined method for the quantification of NETosis ex vivo by flow cytometry and live cell imaging. A neutrophil elastase-activated fluorescent probe is combined with membrane-permeable and impermeable DNA-binding dyes, allowing for the detection of the different stages of NETosis, such as nuclear swelling, the release of neutrophil elastase and extracellular DNA, and loss of intact nuclei. Using this method, we were able to detect a time-dependent increase in the number of neutrophils undergoing NETosis upon stimulation, while NETosis was not detected in untreated neutrophils. Our rapid, simple protocol reduces hands-on time and maximizes reproducibility. This assay is compatible with flow cytometry, facilitating high-throughput detection and quantification of NETosis in purified or mixed cell populations. This assay provides detailed morphological information and visualization of the distinct stages of NETosis while enabling discrimination between live cells, cells that have undergone NETosis, and dead or apoptotic cells. Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

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Neutrophil Extracellular Traps

Concepts

Neutrophil extracellular traps Flow cytometry Ex vivo Cell biology Programmed cell death Elastase Live cell imaging Neutrophil elastase Apoptosis Immune system Extracellular Innate immune system Biology Cytometry In vivo Cell Chemistry Myeloperoxidase Molecular biology Immunology Jurkat cells Single-cell analysis T cell Pancreatic elastase Respiratory burst

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Type: article
Language: en
Landing Page: https://doi.org/10.1093/jimmun/vkaf283.367
PDF: https://academic.oup.com/jimmunol/article-pdf/214/Supplement_1/vkaf283.367/65413729/vkaf283.367.pdf
OA Status: bronze
OpenAlex ID: https://openalex.org/W7106278365

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DOI: https://doi.org/10.1093/jimmun/vkaf283.367

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Title: A sensitive, fast, and efficient method for studying NETosis ex vivo by flow cytometry and live cell imaging 2450

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Type: article

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Language: en

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Publication year: 2025

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Publication date: 2025-11-01

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Authors: Susannah H. Kassmer, Anagha Divekar, Xifeng Yang

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Landing page: https://doi.org/10.1093/jimmun/vkaf283.367

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Concepts: Neutrophil extracellular traps, Flow cytometry, Ex vivo, Cell biology, Programmed cell death, Elastase, Live cell imaging, Neutrophil elastase, Apoptosis, Immune system, Extracellular, Innate immune system, Biology, Cytometry, In vivo, Cell, Chemistry, Myeloperoxidase, Molecular biology, Immunology, Jurkat cells, Single-cell analysis, T cell, Pancreatic elastase, Respiratory burst

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abstract_inverted_index.release	112
abstract_inverted_index.Abstract	0
abstract_inverted_index.Cellular	220
abstract_inverted_index.Defense:	219
abstract_inverted_index.NETosis,	6, 106, 206
abstract_inverted_index.allowing	97
abstract_inverted_index.combined	90
abstract_inverted_index.detailed	185
abstract_inverted_index.detected	148
abstract_inverted_index.distinct	192
abstract_inverted_index.elastase	23, 115
abstract_inverted_index.enabling	197
abstract_inverted_index.hands-on	157
abstract_inverted_index.imaging.	83
abstract_inverted_index.increase	134
abstract_inverted_index.multiple	41
abstract_inverted_index.protocol	155
abstract_inverted_index.provides	184
abstract_inverted_index.purified	177
abstract_inverted_index.research	60
abstract_inverted_index.urgently	53
abstract_inverted_index.Responses	216
abstract_inverted_index.analyzing	47
abstract_inverted_index.apoptotic	210
abstract_inverted_index.chromatin	14
abstract_inverted_index.cytometry	79
abstract_inverted_index.decorated	16
abstract_inverted_index.detection	100, 171
abstract_inverted_index.different	103
abstract_inverted_index.diseases,	42
abstract_inverted_index.eliminate	31
abstract_inverted_index.including	21
abstract_inverted_index.maximizes	160
abstract_inverted_index.measuring	49
abstract_inverted_index.sensitive	66
abstract_inverted_index.swelling,	110
abstract_inverted_index.undergone	205
abstract_inverted_index.untreated	150
abstract_inverted_index.Categories	213
abstract_inverted_index.Mechanisms	221
abstract_inverted_index.compatible	165
abstract_inverted_index.cytometry,	168
abstract_inverted_index.neutrophil	9, 22, 85, 114
abstract_inverted_index.pathogens.	32
abstract_inverted_index.proteases,	20
abstract_inverted_index.undergoing	140
abstract_inverted_index.DNA-binding	95
abstract_inverted_index.Description	1
abstract_inverted_index.advancement	57
abstract_inverted_index.demonstrate	64
abstract_inverted_index.fluorescent	87
abstract_inverted_index.impermeable	94
abstract_inverted_index.information	187
abstract_inverted_index.neutrophils	7, 139
abstract_inverted_index.streamlined	68
abstract_inverted_index.facilitating	169
abstract_inverted_index.neutrophils.	151
abstract_inverted_index.pathogenesis	39
abstract_inverted_index.populations.	181
abstract_inverted_index.standardized	44
abstract_inverted_index.stimulation,	143
abstract_inverted_index.extracellular	10, 117
abstract_inverted_index.morphological	186
abstract_inverted_index.visualization	189
abstract_inverted_index.antimicrobials	18
abstract_inverted_index.discrimination	198
abstract_inverted_index.quantification	72, 173
abstract_inverted_index.time-dependent	133
abstract_inverted_index.high-throughput	170
abstract_inverted_index.reproducibility.	161
abstract_inverted_index.elastase-activated	86
abstract_inverted_index.membrane-permeable	92
cited_by_percentile_year
countries_distinct_count	1
institutions_distinct_count	3
citation_normalized_percentile.value	0.74649116
citation_normalized_percentile.is_in_top_1_percent	False
citation_normalized_percentile.is_in_top_10_percent	False