遗传毒性测试评估化合物造成 DNA 损伤的可能性。有许多遗传毒理学筛选试验旨在评估早期药物开发中化学物质对 DNA 损伤的潜力，帮助识别那些具有低风险潜力的有前景的药物，这些药物会造成导致人类癌症风险的遗传损伤。尽管如此，体外测试会产生大量误导性阳性结果，其后果可能导致不必要的动物测试和/或放弃有希望的候选药物。了解化学作用模式 (MoA) 对于确定物质真正的潜在遗传毒性至关重要，从而将风险转化为临床。在这里，我们展示了一种简单、稳健的方案，用于使用 ɣH2AX、p53 和 pH3S28 抗体以及 DRAQ5™ DNA 染色对固定的、人淋巴细胞 p53 熟练的 TK6 细胞进行染色，从而能够通过成像流式细胞术等显微镜方法分析未裂解的细胞。在这里，我们使用了 Cytek® Amnis® ImageStream®X Mk II，它提供了一个高通量采集平台，具有流式细胞术的灵敏度和与显微镜相关的空间形态信息。使用 ImageStream 制造商的软件 (IDEAS® 6.2)，开发了一种掩蔽策略来自动检测和量化微核事件 (MN) 并表征生物标志物群体。开发的门控策略能够生成能够自动批量处理数据文件的模板，同时量化细胞周期、MN、ɣH2AX、p53 和 pH3 群体。通过这种方式，我们演示了多重系统如何使用成像流式细胞术平台上的未裂解细胞进行 DNA 损伤评估以及 MN 识别。作为概念验证，我们使用工具化学品多菌灵和甲磺酸甲酯 (MMS) 来证明该测定能够使用建立的生物标志物图谱正确识别染色体断裂或非优生 MoA。© 2024。作者。

Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer's software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay's ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.© 2024. The Author(s).