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Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro

Jatin R Verma, Danielle S G Harte, Ume-Kulsoom Shah, Huw Summers Orcid Logo, Catherine A Thornton, Shareen H Doak, Gareth J S Jenkins, Paul Rees Orcid Logo, John W Wills, George Johnson Orcid Logo

Mutagenesis, Volume: 33, Issue: 4, Pages: 283 - 289

Swansea University Authors: Huw Summers Orcid Logo, Paul Rees Orcid Logo, George Johnson Orcid Logo

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DOI (Published version): 10.1093/mutage/gey021

Abstract

Use of imaging flow cytometry to assess induced DNA damage via the cytokinesis block micronucleus (CBMN) assay has thus far been limited to radiation dosimetry in human lymphocytes using high end, ‘ImageStream X’ series imaging cytometers. Its potential to enumerate chemically induced DNA damage usi...

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Published in: Mutagenesis
ISSN: 0267-8357 1464-3804
Published: 2018
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URI: https://cronfa.swan.ac.uk/Record/cronfa45207
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Its potential to enumerate chemically induced DNA damage using in vitro cell lines remains unexplored. In the present manuscript, we investigate the more affordable FlowSight&#xAE; imaging cytometry platform to assess in vitro micronucleus (MN) induction in the human lymphoblastoid TK6 and metabolically competent MCL-5 cells treated with Methyl Methane Sulfonate (MMS) (0&#x2013;5 &#xB5;g/ml), Carbendazim (0&#x2013;1.6 &#xB5;g/ml), and Benzo[a]Pyrene (B[a]P) (0&#x2013;6.3 &#xB5;g/ml) for a period of 1.5&#x2013;2 cell-cycles. Cells were fixed, and nuclei and MN were stained using the fluorescent nuclear dye DRAQ5&#x2122;. Image acquisition was carried out using a 20X objective on a FlowSight&#xAE; imaging cytometer (Amnis, part of Merck Millipore) equipped with a 488 nm laser. Populations of &#x223C;20000 brightfield cell images, alongside DRAQ5&#x2122; stained nuclei/MN were rapidly collected (&#x2264;10 min). Single, in-focus cells suitable for scoring were then isolated using the IDEAS&#xAE; software. An overlay of the brightfield cell outlines and the DRAQ5 nuclear fluorescence was used to facilitate scoring of mono-, bi-, tri-, and tetra-nucleated cells with or without MN events and in context of the cytoplasmic boundary of the parent cell.To establish the potential of the FlowSight&#xAE; platform, and to establish &#x2018;ground truth&#x2019; cell classification for the supervised machine learning based scoring algorithm that represents the next stage of our project, the captured images were scored manually. Alongside, MN frequencies were also derived using the &#x2018;gold standard&#x2019; light microscopy and manual scoring. A minimum of 3000 bi-nucleated cells were assessed using both approaches. Using the benchmark dose approach, the comparability of genotoxic potency estimations for the different compounds and cell lines was assessed across the two scoring platforms as highly similar. This study therefore provides essential proof-of-concept that FlowSight&#xAE; imaging cytometry is capable of reproducing the results of &#x2018;gold standard&#x2019; manual scoring by light microscopy. 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spelling 2018-11-05T10:44:03.4285405 v2 45207 2018-10-25 Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro a61c15e220837ebfa52648c143769427 0000-0002-0898-5612 Huw Summers Huw Summers true false 537a2fe031a796a3bde99679ee8c24f5 0000-0002-7715-6914 Paul Rees Paul Rees true false 37d0f121db69fd09f364df89e4405e31 0000-0001-5643-9942 George Johnson George Johnson true false 2018-10-25 MEDE Use of imaging flow cytometry to assess induced DNA damage via the cytokinesis block micronucleus (CBMN) assay has thus far been limited to radiation dosimetry in human lymphocytes using high end, ‘ImageStream X’ series imaging cytometers. Its potential to enumerate chemically induced DNA damage using in vitro cell lines remains unexplored. In the present manuscript, we investigate the more affordable FlowSight® imaging cytometry platform to assess in vitro micronucleus (MN) induction in the human lymphoblastoid TK6 and metabolically competent MCL-5 cells treated with Methyl Methane Sulfonate (MMS) (0–5 µg/ml), Carbendazim (0–1.6 µg/ml), and Benzo[a]Pyrene (B[a]P) (0–6.3 µg/ml) for a period of 1.5–2 cell-cycles. Cells were fixed, and nuclei and MN were stained using the fluorescent nuclear dye DRAQ5™. Image acquisition was carried out using a 20X objective on a FlowSight® imaging cytometer (Amnis, part of Merck Millipore) equipped with a 488 nm laser. Populations of ∼20000 brightfield cell images, alongside DRAQ5™ stained nuclei/MN were rapidly collected (≤10 min). Single, in-focus cells suitable for scoring were then isolated using the IDEAS® software. An overlay of the brightfield cell outlines and the DRAQ5 nuclear fluorescence was used to facilitate scoring of mono-, bi-, tri-, and tetra-nucleated cells with or without MN events and in context of the cytoplasmic boundary of the parent cell.To establish the potential of the FlowSight® platform, and to establish ‘ground truth’ cell classification for the supervised machine learning based scoring algorithm that represents the next stage of our project, the captured images were scored manually. Alongside, MN frequencies were also derived using the ‘gold standard’ light microscopy and manual scoring. A minimum of 3000 bi-nucleated cells were assessed using both approaches. Using the benchmark dose approach, the comparability of genotoxic potency estimations for the different compounds and cell lines was assessed across the two scoring platforms as highly similar. This study therefore provides essential proof-of-concept that FlowSight® imaging cytometry is capable of reproducing the results of ‘gold standard’ manual scoring by light microscopy. We conclude that, with the right automated scoring algorithm, imaging flow cytometry could revolutionise the reportability and scoring throughput of the CBMN assay. Journal Article Mutagenesis 33 4 283 289 0267-8357 1464-3804 cell cycle, flow cytometry, alkanesulfonates, benchmarking, cell lines, cell nucleus, cytoplasm, dna damage, dyes, fluorescence, immunoglobulins, thyroid-stimulating, infectious mononucleosis, lasers, lymphocytes, methane, micronucleus, pyrenes, radiometry, software, diagnostic imaging, tetracycline, cytokinesis, gold standard, light microscopy, supervised machine learning, proof of concept studies 11 10 2018 2018-10-11 10.1093/mutage/gey021 COLLEGE NANME Biomedical Engineering COLLEGE CODE MEDE Swansea University 2018-11-05T10:44:03.4285405 2018-10-25T09:01:35.7564873 Faculty of Science and Engineering School of Engineering and Applied Sciences - Uncategorised Jatin R Verma 1 Danielle S G Harte 2 Ume-Kulsoom Shah 3 Huw Summers 0000-0002-0898-5612 4 Catherine A Thornton 5 Shareen H Doak 6 Gareth J S Jenkins 7 Paul Rees 0000-0002-7715-6914 8 John W Wills 9 George Johnson 0000-0001-5643-9942 10 0045207-25102018090816.pdf verma2018v2.pdf 2018-10-25T09:08:16.4800000 Output 669608 application/pdf Accepted Manuscript true 2019-09-11T00:00:00.0000000 true eng
title Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro
spellingShingle Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro
Huw Summers
Paul Rees
George Johnson
title_short Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro
title_full Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro
title_fullStr Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro
title_full_unstemmed Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro
title_sort Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro
author_id_str_mv a61c15e220837ebfa52648c143769427
537a2fe031a796a3bde99679ee8c24f5
37d0f121db69fd09f364df89e4405e31
author_id_fullname_str_mv a61c15e220837ebfa52648c143769427_***_Huw Summers
537a2fe031a796a3bde99679ee8c24f5_***_Paul Rees
37d0f121db69fd09f364df89e4405e31_***_George Johnson
author Huw Summers
Paul Rees
George Johnson
author2 Jatin R Verma
Danielle S G Harte
Ume-Kulsoom Shah
Huw Summers
Catherine A Thornton
Shareen H Doak
Gareth J S Jenkins
Paul Rees
John W Wills
George Johnson
format Journal article
container_title Mutagenesis
container_volume 33
container_issue 4
container_start_page 283
publishDate 2018
institution Swansea University
issn 0267-8357
1464-3804
doi_str_mv 10.1093/mutage/gey021
college_str Faculty of Science and Engineering
hierarchytype
hierarchy_top_id facultyofscienceandengineering
hierarchy_top_title Faculty of Science and Engineering
hierarchy_parent_id facultyofscienceandengineering
hierarchy_parent_title Faculty of Science and Engineering
department_str School of Engineering and Applied Sciences - Uncategorised{{{_:::_}}}Faculty of Science and Engineering{{{_:::_}}}School of Engineering and Applied Sciences - Uncategorised
document_store_str 1
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description Use of imaging flow cytometry to assess induced DNA damage via the cytokinesis block micronucleus (CBMN) assay has thus far been limited to radiation dosimetry in human lymphocytes using high end, ‘ImageStream X’ series imaging cytometers. Its potential to enumerate chemically induced DNA damage using in vitro cell lines remains unexplored. In the present manuscript, we investigate the more affordable FlowSight® imaging cytometry platform to assess in vitro micronucleus (MN) induction in the human lymphoblastoid TK6 and metabolically competent MCL-5 cells treated with Methyl Methane Sulfonate (MMS) (0–5 µg/ml), Carbendazim (0–1.6 µg/ml), and Benzo[a]Pyrene (B[a]P) (0–6.3 µg/ml) for a period of 1.5–2 cell-cycles. Cells were fixed, and nuclei and MN were stained using the fluorescent nuclear dye DRAQ5™. Image acquisition was carried out using a 20X objective on a FlowSight® imaging cytometer (Amnis, part of Merck Millipore) equipped with a 488 nm laser. Populations of ∼20000 brightfield cell images, alongside DRAQ5™ stained nuclei/MN were rapidly collected (≤10 min). Single, in-focus cells suitable for scoring were then isolated using the IDEAS® software. An overlay of the brightfield cell outlines and the DRAQ5 nuclear fluorescence was used to facilitate scoring of mono-, bi-, tri-, and tetra-nucleated cells with or without MN events and in context of the cytoplasmic boundary of the parent cell.To establish the potential of the FlowSight® platform, and to establish ‘ground truth’ cell classification for the supervised machine learning based scoring algorithm that represents the next stage of our project, the captured images were scored manually. Alongside, MN frequencies were also derived using the ‘gold standard’ light microscopy and manual scoring. A minimum of 3000 bi-nucleated cells were assessed using both approaches. Using the benchmark dose approach, the comparability of genotoxic potency estimations for the different compounds and cell lines was assessed across the two scoring platforms as highly similar. This study therefore provides essential proof-of-concept that FlowSight® imaging cytometry is capable of reproducing the results of ‘gold standard’ manual scoring by light microscopy. We conclude that, with the right automated scoring algorithm, imaging flow cytometry could revolutionise the reportability and scoring throughput of the CBMN assay.
published_date 2018-10-11T03:56:54Z
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