Development of an In Vitro whole cell Micronucleus Multiplex expansion assay: Using DRAQ5™ and the DNA damage biomarkers ɣH2AX, P53, pH3 along with cell cycle relationships and the MN genotoxic endpoint to assess chemical Mode of Action on an imaging flow cytometry platform with template development

Genetic toxicity testing is the assessment of compounds, and their respective metabolites, potential to cause DNA damage either directly or indirectly. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development to help identify promising drugs that have a low risk potential of causing damage leading to cancer in humans. Despite this, in vitro tests generate a high number of miss-leading positives, the consequences of which lead to unnecessary animal testing and abandoning promising drug candidates. Understanding chemical Mode of Action (MoA) is vital in identifying true genotoxic potential of substances. Here I demonstrate a simple robust protocol for optimised staining of fixed human lymphoblast P53 proficient TK6 cells with the antibodies; Anti-ɣH2AX, Anti-P53 and Ant-pH3S28 along with DRAQ5™ DNA staining in a whole cell multiplex system that is suitable for analysis via microscopy or imaging flow cytometry. Use of the Amnis FlowSight® and ImageStream X Mark II® platform provided a high content high throughput acquisition platform with the sensitivity of flow cytometry and accuracy of image analysis. Using both optimal and suboptimal lasers for fluorophore excitation demonstrated that a multiplex system for DNA damage assessment including MN was possible in un-lysed cells. IDEAS 6.2 template generation allowed for batch processing of data samples extracting the following metrics: Cell Cycle, Micronucleus (MN), ɣH2AX, P53, PH3, G1 ɣH2AX, G1 P53, S ɣH2AX, S P53, G2 ɣH2AX, G2/M P53, Prophase, Metaphase, Anaphase and Abnormal mitosis. Furthermore, high content nature of the platform using imagery allows for identification of rare cellular event assessment particularly preliminary data on biomarker signal found within MN. The system found differences in the biomarker metric responses between the chemicals; MMS, Carbendazim, ARA-C, Vinblastine, Etoposide and Crizotinib suggesting potential for chemical MoA elucidation.