A multi-biomarker micronucleus assay using imaging flow cytometry

Harte, Danielle; Lynch, Anthony; Verma, Jatin; Rees, Paul; Filby, Andrew; Wills, John W.; Johnson, George

doi:10.1007/s00204-024-03801-7

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A multi-biomarker micronucleus assay using imaging flow cytometry

Danielle Harte, Anthony Lynch, Jatin Verma, Paul Rees

, Andrew Filby, John W. Wills, George Johnson

Archives of Toxicology, Volume: 98, Pages: 3137 - 3153

Swansea University Authors: Danielle Harte, Anthony Lynch, Jatin Verma, Paul Rees , George Johnson

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DOI (Published version): 10.1007/s00204-024-03801-7

Abstract

Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for caus...

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Published in:	Archives of Toxicology
ISSN:	0340-5761 1432-0738
Published:	Springer Science and Business Media LLC 2024
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URI:	https://cronfa.swan.ac.uk/Record/cronfa66744
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spelling	v2 66744 2024-06-17 A multi-biomarker micronucleus assay using imaging flow cytometry 183eddc613937f235a28c10f63079678 Danielle Harte Danielle Harte true false 94e539dab2511bf7fa596450e5cadabf Anthony Lynch Anthony Lynch true false 6c11cec3815148d928211f6648a6dce9 Jatin Verma Jatin Verma true false 537a2fe031a796a3bde99679ee8c24f5 0000-0002-7715-6914 Paul Rees Paul Rees true false 37d0f121db69fd09f364df89e4405e31 0000-0001-5643-9942 George Johnson George Johnson true false 2024-06-17 MEDS Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer’s software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay’s ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established. Journal Article Archives of Toxicology 98 3137 3153 Springer Science and Business Media LLC 0340-5761 1432-0738 ImageStream; Micronucleus; NAM; DNA damage; MoA; Biomarker 1 9 2024 2024-09-01 10.1007/s00204-024-03801-7 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University SU Library paid the OA fee (TA Institutional Deal) The authors acknowledge and thank the Life Science Bridging Fund within the Life Science Research Network Wales (LSBF/R3-007) and Swansea University for providing funding in support of the authors PhD. We also acknowledge UK Biotechnology and Biological Sciences Research Council (BB/P026818/1) for supporting this work. 2024-11-01T15:24:53.0542543 2024-06-17T16:26:12.6720161 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science Danielle Harte 1 Anthony Lynch 2 Jatin Verma 3 Paul Rees 0000-0002-7715-6914 4 Andrew Filby 5 John W. Wills 6 George Johnson 0000-0001-5643-9942 7 66744__30947__05a08ffa17de4467906ffed1be2dfc74.pdf 66744.VoR.pdf 2024-07-23T13:56:44.9066104 Output 1988587 application/pdf Version of Record true © The Author(s) 2024. This article is licensed under a Creative Commons Attribution 4.0 International License. true eng http://creativecommons.org/licenses/by/4.0/
title	A multi-biomarker micronucleus assay using imaging flow cytometry
spellingShingle	A multi-biomarker micronucleus assay using imaging flow cytometry Danielle Harte Anthony Lynch Jatin Verma Paul Rees George Johnson
title_short	A multi-biomarker micronucleus assay using imaging flow cytometry
title_full	A multi-biomarker micronucleus assay using imaging flow cytometry
title_fullStr	A multi-biomarker micronucleus assay using imaging flow cytometry
title_full_unstemmed	A multi-biomarker micronucleus assay using imaging flow cytometry
title_sort	A multi-biomarker micronucleus assay using imaging flow cytometry
author_id_str_mv	183eddc613937f235a28c10f63079678 94e539dab2511bf7fa596450e5cadabf 6c11cec3815148d928211f6648a6dce9 537a2fe031a796a3bde99679ee8c24f5 37d0f121db69fd09f364df89e4405e31
author_id_fullname_str_mv	183eddc613937f235a28c10f63079678_*_Danielle Harte 94e539dab2511bf7fa596450e5cadabf__Anthony Lynch 6c11cec3815148d928211f6648a6dce9__Jatin Verma 537a2fe031a796a3bde99679ee8c24f5__Paul Rees 37d0f121db69fd09f364df89e4405e31_**_George Johnson
author	Danielle Harte Anthony Lynch Jatin Verma Paul Rees George Johnson
author2	Danielle Harte Anthony Lynch Jatin Verma Paul Rees Andrew Filby John W. Wills George Johnson
format	Journal article
container_title	Archives of Toxicology
container_volume	98
container_start_page	3137
publishDate	2024
institution	Swansea University
issn	0340-5761 1432-0738
doi_str_mv	10.1007/s00204-024-03801-7
publisher	Springer Science and Business Media LLC
college_str	Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id	facultyofmedicinehealthandlifesciences
hierarchy_top_title	Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id	facultyofmedicinehealthandlifesciences
hierarchy_parent_title	Faculty of Medicine, Health and Life Sciences
department_str	Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
document_store_str	1
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description	Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer’s software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay’s ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.
published_date	2024-09-01T15:24:50Z
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score	11.037603

A multi-biomarker micronucleus assay using imaging flow cytometry

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