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The In Vitro Micronucleus Assay and FISH Analysis

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Genotoxicity and DNA Repair

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

The cytokinesis-block micronucleus cytome (CBMN-cyt) assay was originally established as an ideal system for evaluating chromosomal damage in terms of micronuclei formation. Throughout the years, the micronucleus assay evolved in a comprehensive system for assessing cytogenetic damage, cytostasis and cytotoxicity. The CBMN-cyt assay in peripheral blood lymphocytes and in other cultured mammalian cells is the most common approach to evaluate chromosomal damage induced by environmental agents, including emerging compounds as nanomaterials, and it is the most frequent test system in biomonitoring human populations.

When coupled with fluorescence in situ hybridization (FISH), CBMN-cyt assay is able to reveal the capability to induce structural chromosome aberrations (clastogenic activity) and/or numerical chromosome changes (aneuploidogenic activity).

The methods for CBMN-cyt assay and FISH described here refer to the use of separate lymphocytes and whole blood cultures involving the block of cytokinesis with cytochalasin B (cyt-B) but other cell systems of different origin can be successfully used. This chapter describes in details well-established protocols for sample processing, slide preparation and scoring criteria.

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Acknowledgements

SDB and CU are granted by the FP7 project No 280716, SANOWORK (www.sanowork.eu). We would like to acknowledge Davide Tesoro for drawing figures.

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Authors and Affiliations

  1. Department of Translational Research and New Technologies in Medicine and Surgery, Medical Genetics Unit, University of Pisa, Pisa, Italy

    Lucia Migliore, Sebastiano Di Bucchianico & Chiara Uboldi

Authors
  1. Lucia Migliore
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  2. Sebastiano Di Bucchianico
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  3. Chiara Uboldi
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Corresponding author

Correspondence to Lucia Migliore .

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Editors and Affiliations

  1. Universidad de Oviedo, Oviedo, Spain

    L. María Sierra

  2. Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal

    Isabel Gaivão

Glossary

BN

Binucleated cells

BNMN

Binucleated micronucleated cells

BrdU

Bromodeoxyuridine

CBMN

Cytokinesis-block micronucleus

CBMN-cyt

Cytokinesis-block micronucleus cytome

CBPI

Cytokinesis-block proliferation index

CENP-A

Centromere protein A

CENP-B

Centromere protein B

cyt-B

Cytochalasin-B

DAPI

4′,6-Diamidin-2-fenilindolo

DMSO

Dimethylsulfoxide

DNA

Deoxyribonucleic acid

FBS

Foetal bovine serum

FISH

Fluorescence in situ hybridization

HBSS

Hank’s balanced salt solution

MN

Micronucleus/i

MN C

Micronucleus/i centromere negative

MN C+

Micronucleus/i centromere positive

NBUD

Nuclear bud/s

NM

Nanomaterial/s

NPB

Nucleoplasmic bridge/s

PBS

Phosphate-buffered solution

PHA

Phytohemagglutinin

PI

Proliferation index

RICC

Relative increase in cell counts

RT

Room temperature

SSC

Sodium chloride/sodium citrate

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Migliore, L., Di Bucchianico, S., Uboldi, C. (2014). The In Vitro Micronucleus Assay and FISH Analysis. In: Sierra, L., Gaivão, I. (eds) Genotoxicity and DNA Repair. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1068-7_5

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  • DOI: https://doi.org/10.1007/978-1-4939-1068-7_5

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