In vitro micronucleus scoring by flow cytometry: Differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability
- 22 September 2005
- journal article
- research article
- Published by Wiley in Environmental and Molecular Mutagenesis
- Vol. 47 (1), 56-66
- https://doi.org/10.1002/em.20170
Abstract
The in vitro micronucleus test has received considerable attention in recent years for its use in drug safety assessment and toxicological research. The less tedious nature of the assay relative to chromosome aberration analyses is a driving force, and explains why many chemical and drug safety programs have adopted the endpoint. Development of a high-throughput micronucleus scoring system would further enhance the utility of the assay for lead optimization and other early drug development work. Although several variations of a flow cytometric (FCM) method for scoring cell-culture-derived micronuclei (MN) have been described in the literature, they have been unable to distinguish true MN from apoptotic and necrotic chromatin (Nüsse M and Marx K 1997 : Mutat Res 392: 109–115). Here, we report advances to this methodology whereby a sequential staining procedure is used to differentially label these types of sub-2n particles. With the use of ethidium monoazide (EMA), the chromatin of dead and dying cells is labeled. After a photoactivation step that covalently binds EMA to chromatin, cytoplasmic membranes are digested and resulting lysates are incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and sub-2n particles that are labeled with either SYTOX or SYTOX and EMA. Preliminary studies with heat-shocked L5178Y mouse cells demonstrated that EMA stains necrotic and mid- through late-stage apoptotic cells. Importantly, the sequential labeling procedure provided reliable micronucleus enumeration, even when cultures contained high percentages of dead cells. Subsequently, experiments with the following diverse genotoxicants were performed: hydroxyurea, methyl methanesulfonate, benzo[a]pyrene, etoposide, cyclophosphamide, and vinblastine. Additionally, the nongenotoxicants sucrose, tributyltin methoxide, and dexamethasone were tested up to 5 mg/ ml, or to cytotoxic concentrations. FCM data were found to correspond closely with microscopy-based measurements. Collectively, these data suggest that this sequential EMA/SYTOX staining procedure provides reliable, high-throughput enumeration of in vitro MN. Environ. Mol. Mutagen., 2006.Keywords
This publication has 29 references indexed in Scilit:
- Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay: position of the GUM working group on the in vitro micronucleus testMutation Research, 1998
- Cytometry in cell necrobiology: Analysis of apoptosis and accidental cell death (necrosis)Cytometry, 1997
- Image processing algorithms for the automated micronucleus assay in binucleated human lymphocytesCytometry, 1995
- Flow cytometric detection of micronuclei by combined staining of DNA and membranesCytometry, 1995
- Flow Cytometric Analysis of Micronuclei in the CD2 ± Subpopulation of Human Lymphocytes Enriched by Magnetic SeparationInternational Journal of Radiation Biology, 1995
- Scoring of radiation-induced microuclei in cytokinesis-blocked human lymphocytes by automated image analysisCytometry, 1994
- The in vitro cytokinesis-block micronucleus assay: a detailed description of an improved slide preparation technique for the automated detection of micronuclei in human lymphocytesMutagenesis, 1994
- Multiparametric flow cytometric analysis of radiation‐induced micronuclei in mammalian cell culturesCytometry, 1992
- Flow cytometric analysis of micronuclei found in cells after irradiationCytometry, 1984