2005
DOI: 10.1002/em.20170
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In vitro micronucleus scoring by flow cytometry: Differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability

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 sever… Show more

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Cited by 140 publications
(111 citation statements)
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“…The high numbers of cells captured by the ISX illustrate the improvement in statistical robustness that can be achieved in comparison to microscope‐based versions of the assay. In addition, conventional flow cytometry methods that do not use Cyt‐B typically analyze between 20,000 and 40,000 lysed nuclei 30, 31, 61. The number of MONO cells scored from samples treated without Cyt‐B using the ISX method fall roughly in the middle of this range and have the added benefit of associated BF and fluorescent imagery for every cell collected.…”
Section: Discussionmentioning
confidence: 99%
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“…The high numbers of cells captured by the ISX illustrate the improvement in statistical robustness that can be achieved in comparison to microscope‐based versions of the assay. In addition, conventional flow cytometry methods that do not use Cyt‐B typically analyze between 20,000 and 40,000 lysed nuclei 30, 31, 61. The number of MONO cells scored from samples treated without Cyt‐B using the ISX method fall roughly in the middle of this range and have the added benefit of associated BF and fluorescent imagery for every cell collected.…”
Section: Discussionmentioning
confidence: 99%
“…The speed of this method and the need for minimal user intervention are attractive features for toxicology testing. Over the last decade, significant improvements were made to the methodology by Avlasevich et al 30 and Bryce et al 31 to incorporate additional fluorescent dyes in order to remedy the difficulties encountered when differentiating MN, cellular debris, and free chromosomes from mitotic cells. This method was successful and configured into a commercially available kit by Litron Laboratories and several publications employing many well‐known clastogens and aneugens have demonstrated that statistically significant increases in MN frequency can be detected when compared with controls 32, 33.…”
Section: Introductionmentioning
confidence: 99%
“…Subsequent stripping of cytoplasmic membranes and incubation with the pan-nucleic acid dye SYTOX Green (plus RNase) provides a suspension of free nuclei and micronuclei (MN) that are differentially stained relative to chromatin associated with dead/dying cells. Using this method, good agreement between microscopy-and flow cytometry-based scoring was reported for mouse L5178Y and human TK6 cells [12][13].…”
Section: Introductionmentioning
confidence: 87%
“…The EMA-positive phenotype is acquired upon loss of membrane integrity, and occurs during necrosis, and also mid-to late-stage apoptosis [12]. In the present investigation, the statistic is expressed as the percentage of EMA-positive chromatin as a fraction of all chromatin.…”
Section: 43mentioning
confidence: 95%
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