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Buccal micronucleus cytome assay

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Abstract

The Buccal Micronucleus Cytome (BMCyt) assay is a minimally invasive method for studying DNA damage, chromosomal instability, cell death and the regenerative potential of human buccal mucosal tissue. This method is increasingly used in molecular epidemiological studies for investigating the impact of nutrition, lifestyle factors, genotoxin exposure and genotype on DNA damage, chromosome malsegregation and cell death. The biomarkers measured in this assay have been associated with increased risk of accelerated ageing, cancer and neurodegenerative diseases. This protocol describes one of the current established methods for buccal cell collection using a small-headed toothbrush, the generation of a single-cell suspension, slide preparation using cytocentrifugation, fixation and staining using Feulgen and Light Green for both bright field and fluorescence microscopic analysis. The scoring criteria for micronuclei and other nuclear anomalies are also described in detail. The protocol in its current form takes approximately 4 h to complete from the time of buccal cell collection to the generation of stained slides for microscopic analysis.

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Figure 1: Diagrammatic representation of a cross section of normal buccal mucosa.
Figure 2: Sequential origins and spatio-temporal sequence of the various cell types in the BMCyt assay.
Figure 3: Diagrammatic representation of the various cell types scored in the BMCyt assay.
Figure 4
Figure 5: Images of the different cell types stained using Feulgen and Light Green scored in the BMCyt assay viewed by transmitted light or under fluorescence with a far red filter.
Figure 6

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Change history

  • 22 September 2011

    In the version of this article initially published, in the REAGENTS SETUP section (page 832), the proportions of salts shown in the "Buccal cell buffer" section were incorrect. "1.2 g EDTA and 37.2 g of sodium chloride" should have read "38.0 g EDTA and 1.2 g of sodium chloride".

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Acknowledgements

We are grateful to the numerous volunteers whose donation of buccal cell samples over the many years has enabled us to achieve the refinement of this protocol. We are particularly indebted to Professor Hans Stich who pioneered this field of research and whose vision enabled the evolution of this protocol.

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Correspondence to Philip Thomas or Michael Fenech.

Supplementary information

Supplementary Table 1: Example of a Buccal Micronucleus Cytome assay score sheet.

Diff=Differentiated; BN=Binucleated; PYK=Pyknotic; CC=Condensed Chromatin; KYL=Karyolytic; KHC=Karyorrhectic ; MN = Micronucleus; NBUD = Nuclear Bud; no = number (DOC 32 kb)

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Thomas, P., Holland, N., Bolognesi, C. et al. Buccal micronucleus cytome assay. Nat Protoc 4, 825–837 (2009). https://doi.org/10.1038/nprot.2009.53

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