Abstract
The in vitro micronucleus (MN) assay is a well-established test for evaluating genotoxicity and cytotoxicity. The use of manual microscopy to perform the assay can be laborious and often suffers from user subjectivity and interscorer variability. Automated methods including slide-scanning microscopy and conventional flow cytometry have been developed to eliminate scorer bias and improve throughput. However, these methods possess several limitations such as lack of cytoplasmic visualization using slide-scanning microscopy and the inability to visually confirm the legitimacy of MN or storage of image data for re-evaluation using flow cytometry. The ImageStreamX® MK II (ISX) imaging flow cytometer has been demonstrated to overcome all of these limitations. The ISX combines the speed, statistical robustness, and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery of microscopy and possesses the ability to store all collected image data. This paper details the methodology developed to perform the in vitro MN assay in human lymphoblastoid TK6 cells on the ISX. High resolution images of micronucleated mono- and bi-nucleated cells as well as polynucleated cells can be acquired at a high rate of capture. All images can then be automatically identified, categorized and enumerated in the data analysis software that accompanies the ImageStream, allowing for the scoring of both genotoxicity and cytotoxicity. The results demonstrate that statistically significant increases in MN frequency when compared with solvent controls can be detected at varying levels of cytotoxicity following exposure to well-known aneugens and clastogens. This work demonstrates a fully automated method for performing the in vitro micronucleus assay on the ISX imaging flow cytometry platform. © 2018 The Author. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.
Keywords:
ImageStream®; In vitro micronucleus assay; TK6 cells; automation; cytochalasin B; cytotoxicity; genotoxicity; image analysis; imaging flow cytometry.