PDF(Pubmed)
Abstract：
(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.
摘要：
（1）背景：使用磷酸化组蛋白生物标志物（γH2AX）在体外检测DNA双链断裂是一种越来越流行的体外遗传毒性测量方法，因为它很敏感，具体和适用于高通量分析。通过流式细胞术或显微镜检测γH2AX反应，后者更容易获得。然而,作者很少发布细节，数据,以及总体荧光强度量化的工作流程，这阻碍了可重复性。(2)方法：我们使用戊霉素作为模型的基因毒素，两种细胞系（HeLa和CHO-K1）和用于γH2AX免疫荧光检测的商业试剂盒。使用开源软件ImageJ进行生物图像分析。使用来自DAPI通道的分段核测量平均荧光值,并且结果表示为相对于对照的γH2AX荧光的面积缩放的相对倍数变化。细胞毒性表示为细胞核的相对面积。我们介绍了工作流程，数据,和GitHub上的脚本。（3）结果：通过引入的方法获得的输出与预期结果一致，即，戊霉素对孵育24小时后使用的两种细胞系均具有遗传毒性和细胞毒性。（4）结论：从生物图像分析获得的γH2AX的整体荧光强度似乎是流式细胞术的有希望的替代方法。工作流,数据,和脚本共享对于进一步改进生物图像分析方法至关重要。
