Automation of the Buccal Micronucleus Cytome Assay Using Laser Scanning Cytometry
Zbigniew Darzynkiewicz, Elena Holden, Alberto Orfao, William Telford and Donald Wlodkowic
Faculty of Computing, Health and Science
School of Medical Sciences
Laser scanning cytometry (LSC) can be used to quantify the fluorescence intensity or laser light loss (absorbance) of localized modular targets within nuclear and cytoplasmic structures of cells while maintaining the morphological features of the examined tissue. It was aimed to develop as 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 bio-markers 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 karyolitic cells), the following 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 and 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 a LSC.
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