Multiparametric flow cytometric analysis of radiation‐induced micronuclei in mammalian cell cultures

Schreiber, Georg; Beisker, Wolfgang; Bauchinger, M.; Nüsse, Michael

doi:10.1002/cyto.990130114

Cited by 42 publications

(29 citation statements)

References 23 publications

(25 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Recently, Schreiber et al (14,151 reported on their results with an automated flow cytometer. Their method is based on a separate measurement of the number of BN cells in the cultures and the number of MN in the BN cells.…”

Section: Fenech and Morley (5) Allows Identification Of Cells In Intmentioning

confidence: 99%

Scoring of radiation‐induced microuclei in cytokinesis‐blocked human lymphocytes by automated image analysis

Verhaegen¹,

Vral²,

Seuntjens³

et al. 1994

Cytometry

View full text Add to dashboard Cite

The micronucleus assay in human lymphocytes is, at present, frequently used to assess chromosomal damage caused by ionizing radiation or mutagens. Manual scoring of micronuclei (MN) by trained personnel is very time-consuming, tiring work, and the results depend on subjective interpretation of scoring criteria. More objective scoring can be accomplished only if the test can be automated. Furthermore, an automated system allows scoring of large numbers of cells, thereby increasing the statistical significance of the results. This is of special importance for screening programs for low doses of chromosome-damaging agents.In this paper, the first results of our effort to automate the micronucleus assay with an image-analysis system are represented. The method we used is described in detail, and the results are compared to those of other groups. Our system is able to detect 88% of the binucleated lymphocytes on the slides. The procedure consists of a fully automated localization of binucleated cells and counting of the MN within these cells, followed by a simple and fast manual operation in which the false positives are removed. Preliminary measurements for blood samples irradiated with a dose of 1 Gy X-rays indicate that the automated system can find 89% f 12% of the micronuclei within the binucleated cells compared to a manual screening.

show abstract

Section: Fenech and Morley (5) Allows Identification Of Cells In Intmentioning

confidence: 99%

Scoring of radiation‐induced microuclei in cytokinesis‐blocked human lymphocytes by automated image analysis

Verhaegen¹,

Vral²,

Seuntjens³

et al. 1994

Cytometry

View full text Add to dashboard Cite

show abstract

“…These methods are based on image analysis, laser scanning cytometry, or flow cytometry [10][11][12][13][14][15][16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity

Bryce¹,

Bemis²,

Avlasevich³

et al. 2007

Mutation Research/Genetic Toxicology and Environmental Mutagene

176

164

View full text Add to dashboard Cite

This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [Avlasevich et al., Environ. Molec. Mutagen. 47 (2006) 56-66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30 hrs of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1 Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy-and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed nongenotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.

show abstract

“…We have previously reported on the development of a scoring system that may enhance the utility of a flow cytometric procedure originally described by Nüsse and colleagues [7][8][9][10][11]. Most importantly, the new method includes an ethidium monoazide bromide (EMA) staining step in order to label the chromatin of necrotic and mid/late stage apoptotic cells.…”

Section: Introductionmentioning

confidence: 99%

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Bryce

Avlasevich

Bemis

et al. 2008

Mutation Research/Genetic Toxicology and Environmental Mutagene

View full text Add to dashboard Cite

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [Avlasevich et al., Environ. Molec. Mutagen. 47 (2006) [56][57][58][59][60][61][62][63][64][65][66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei-the genotoxicants mitomycin C, etoposide, and vinblastine, and the non-genotoxicants sucrose, staurosporine, or dexamethasone. The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow ™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 hrs over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy-and flow cytometry-based scoring methods detected concentrationdependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

show abstract

Multiparametric flow cytometric analysis of radiation‐induced micronuclei in mammalian cell cultures

Cited by 42 publications

References 23 publications

Scoring of radiation‐induced microuclei in cytokinesis‐blocked human lymphocytes by automated image analysis

Scoring of radiation‐induced microuclei in cytokinesis‐blocked human lymphocytes by automated image analysis

In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

Contact Info

Product

Resources

About