Protocol for Quantifying γH2AX Foci in Irradiated Cells Using Immunofluorescence and Fiji Software

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.