Deng Lu, Wang Danning, Wu Lingying
Department of Gynecologic Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Bio Protoc. 2025 Aug 20;15(16):e5421. doi: 10.21769/BioProtoc.5421.
Quantification of DNA double-strand breaks (DSBs) is critical for assessing genomic damage and cellular response to stress. γH2AX is a well-established marker for DNA double-strand breaks, but its quantification is often performed manually or semi-quantitatively, lacking standardization and reproducibility. Here, we present a standardized and automated workflow for γH2AX foci quantification in irradiated cells using immunofluorescence and a custom Fiji macro. The protocol includes steps for cell irradiation, immunostaining, image acquisition, and automated foci counting. The protocol is also adaptable to colony-like formations in multi-well plates, extending its utility to clonogenic assays. This protocol enables high-throughput, reproducible quantification of DNA damage with minimal user bias and can be readily implemented in routine laboratory settings. Key features • Provides an automated Fiji macro for high-throughput quantification of nuclear γH2AX fluorescence foci with single-nucleus resolution. • Standardized workflow optimized for reproducibility and cross-sample consistency in DSBs detection. • Applicable to nuclear fluorescence foci, as well as colony-like structures in multi-well formats for DNA damage and clonogenic assays.
DNA双链断裂（DSB）的定量分析对于评估基因组损伤和细胞对应激的反应至关重要。γH2AX是一种公认的DNA双链断裂标志物，但其定量分析通常是手动或半定量进行的，缺乏标准化和可重复性。在此，我们提出了一种标准化的自动化工作流程，用于使用免疫荧光和自定义的Fiji宏对受辐照细胞中的γH2AX焦点进行定量分析。该方案包括细胞辐照、免疫染色、图像采集和自动焦点计数等步骤。该方案还适用于多孔板中的集落样形成，将其应用扩展到克隆形成试验。该方案能够以最小的用户偏差实现DNA损伤的高通量、可重复定量分析，并且可以在常规实验室环境中轻松实施。关键特性 • 提供一个自动化的Fiji宏，用于以单细胞核分辨率对细胞核γH2AX荧光焦点进行高通量定量分析。 • 标准化的工作流程，针对DSB检测中的可重复性和跨样本一致性进行了优化。 • 适用于细胞核荧光焦点，以及用于DNA损伤和克隆形成试验的多孔板中的集落样结构。
