Background

Histone H2AX plays essential roles in nucleosome assembly, chromatin remodeling, and DNA repair [1]. DSBs can be induced by various sources, including ionizing radiation, chemical agents (such as etoposide, doxorubicin, and camptothecin), and endogenous biological processes (such as replication stress). Upon induction of DSBs, H2AX is rapidly phosphorylated at serine 139 to form γH2AX [2]. This phosphorylated histone marks the sites of double-strand breaks and recruits cell cycle checkpoint proteins and DNA repair factors to the damaged regions. Since γH2AX levels rise within minutes after double-strand breaks, it is widely used as a biomarker for DNA damage [1]. Quantitative detection of γH2AX expression can be employed to assess the extent of DNA damage [3].

Currently, the quantification of nuclear fluorescence foci such as γH2AX is predominantly performed using manual or semi-quantitative approaches. Manual counting is labor-intensive and subject to observer bias. Some studies adopt intensity-based or area-based metrics as surrogates for foci number, but these lack spatial resolution and fail to distinguish overlapping or clustered foci. Moreover, many published studies do not explicitly describe how foci are quantified, posing challenges to reproducibility and cross-study comparisons.

To address these limitations, we present an automated, reproducible workflow that performs direct foci counting at the single-nucleus level using a custom Fiji macro. Fiji, a widely adopted open-source platform for biomedical image analysis, supports a wide range of file formats (e.g., TIFF, ND2, CZI) and provides robust tools for fluorescence analysis, morphological quantification, 3D reconstruction, and workflow automation through customizable macros and scripting [4,5]. Our protocol leverages Fiji’s core capabilities while maintaining a user-friendly design that requires little coding experience, making it readily accessible to researchers with diverse technical backgrounds.

This protocol offers a complete and streamlined workflow encompassing sample preparation, immunostaining, image preprocessing, and Fiji macro-based quantification. The approach enables nucleus-by-nucleus quantification of fluorescence foci across experimental groups. It is demonstrated using irradiated C-33A cells, with or without siRNA-mediated knockdown of the target gene, allowing assessment of gene-specific effects on cellular radiosensitivity.

Importantly, beyond γH2AX foci quantification, the macro’s underlying image analysis framework is broadly applicable to other counting tasks based on morphologically similar, segmentable regions of interest (ROIs). For example, it can be used to count colony-like structures in multi-well plate-based assays, including clonogenic assays. This extended applicability underscores the macro’s versatility, enabling a unified and user-friendly workflow for high-throughput quantification of structured, ROI-based image features across diverse experimental settings.