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KMID : 1177720180120010033
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Journal of Alternatives to Animal Experiments
2018 Volume.12 No. 1 p.33 ~ p.39
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Three Dimensional Nucleus Imaging analysis & in vitro Genotoxicity Test through Advanced Cytokinesis-Block Micronucleus Assay
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Kim Hye-Ji
Seo In-Cheol
Lee Chae-Eun
Chung Won-Seok
Lee Hyun-Su
Park Sun-Hyun
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Abstract
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The cytokinesis-block micronucleus cytome assay is a measuring of DNA damage in once-divided binucleated (BN) cells scoring micronuclei (MNi) that are widely used as biomarkers of cancer risk in humans on drugs or chemicals. However, this assay is still challenging due to the speed of sample processing and the toxicity of organic chemicals on researcher. To address these problems, we developed a new and convenient method for the micronucleus cytome assay. This method by DAPI stained micronucleus detection is non-toxic to the researcher and is being simplified because it does not require the use of methanol and acetic acid for fixing cell. Especially, three dimensional imaging of nucleus by formalin fixed DAPI staining provides more detailed information for testing the effects of chemical and bio-materials, compared to the classic method. In this study, we examine the spatial distribution of the TANNylated GFPs by advanced cytokinesis-block micronucleus (ACBMN) assay. The TANNylated GFPs were precisely observed in the inner nucleus by three dimensional imaging, whereas GFPs were not detected in any regions of cells. This micronucleus assay can readily be used in genetic based toxicity and efficacy assay to the various bio-chemicals and pharmaceutical evaluation.
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KEYWORD
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The cytokinesis-block micronucleus cytome assay, micronuclei, MNi, advanced cytokinesis-block micronucleus, ACBMN, assay DAPI staining, TANNylated GFPs, Paraformaldehyde, PFA, Three dimensional image
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