title: Automation of the buccal micronucleus cytome assay using laser scanning cytometry.
abstract: Laser scanning cytometry (LSC) can be used to quantify the fluorescence intensity or laser light loss (absorbance) of localized molecular targets within nuclear and cytoplasmic structures of cells while maintaining the morphological features of the examined tissue. It was aimed to develop an automated LSC protocol to study cellular and nuclear anomalies and DNA damage events in human buccal mucosal cells. Since the buccal micronucleus cytome assay has been used to measure biomarkers of DNA damage (micronuclei and/or nuclear buds), cytokinesis defects (binucleated cells), proliferative potential (basal cell frequency), and/or cell death (condensed chromatin, karyorrhexis, and pyknotic and karyolytic cells), the following automated LSC protocol describes scoring criteria for these same parameters using an automated imaging LSC. In this automated LSC assay, cells derived from the buccal mucosa were harvested from the inside of patient's mouths using a small-headed toothbrush. The cells were washed to remove any debris and/or bacteria, and a single-cell suspension prepared and applied to a microscope slide using a cytocentrifuge. Cells were fixed and stained with Feulgen and Light Green stain allowing both chromatic and fluorescent analysis to be undertaken simultaneously with the use of an LSC.
authors.affiliation: CSIRO Food and Nutritional Sciences, Genome Health Nutrigenomics, Adelaide, SA, Australia.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Leifert
authors.forename: Maxime
authors.initials: M
authors.lastname: François
authors.forename: Philip
authors.initials: P
authors.lastname: Thomas
authors.forename: Ed
authors.initials: E
authors.lastname: Luther
authors.forename: Elena
authors.initials: E
authors.lastname: Holden
authors.forename: Michael
authors.initials: M
authors.lastname: Fenech
mesh_terms: D000368:Aged; D000544:Alzheimer Disease / Q000175:diagnosis; D057205:Automation, Laboratory / Q000379:methods*; D015415:Biomarkers; D002469:Cell Separation / Q000379:methods; D004314:Down Syndrome / Q000235:genetics / Q000473:pathology; D004847:Epithelial Cells / Q000473:pathology; D006801:Humans; D007620:Karyometry / Q000379:methods; D047569:Laser Scanning Cytometry / Q000379:methods*; D015162:Micronucleus Tests / Q000379:methods*; D009061:Mouth Mucosa / Q000473:pathology*; D059010:Single-Cell Analysis / Q000379:methods; D055815:Young Adult
chemical_list: D015415:Biomarkers