Journal article 189 views 23 downloads
A multi-biomarker micronucleus assay using imaging flow cytometry
,
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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© The Author(s) 2024. This article is licensed under a Creative Commons Attribution 4.0 International License.
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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...
| Published in: | Archives of Toxicology |
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| ISSN: | 0340-5761 1432-0738 |
| Published: |
Springer Science and Business Media LLC
2024
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| Online Access: |
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| URI: | https://cronfa.swan.ac.uk/Record/cronfa66744 |
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| 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 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. |
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| Keywords: |
ImageStream; Micronucleus; NAM; DNA damage; MoA; Biomarker |
| College: |
Faculty of Medicine, Health and Life Sciences |
| Funders: |
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. |
| Start Page: |
3137 |
| End Page: |
3153 |