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

Area of Science:

• Cell Biology
• Genomics
• Molecular Biology

Background:

• Quantifying DNA double-strand breaks (DSBs) is crucial for understanding genomic damage and cellular stress responses.
• The marker γH2AX is widely used for DSBs, but current quantification methods lack standardization and reproducibility.
• Manual or semi-quantitative analysis introduces user bias and limits throughput.

Purpose of the Study:

• To develop a standardized and automated workflow for quantifying γH2AX foci in irradiated cells.
• To improve the reproducibility and reduce user bias in DNA double-strand break detection.
• To create a high-throughput method adaptable for both nuclear foci and colony-like structures.

Main Methods:

• Development of a custom Fiji macro for automated γH2AX foci counting.
• Implementation of a standardized protocol including cell irradiation, immunofluorescence staining, and image acquisition.
• Adaptation of the workflow for multi-well plate formats, including clonogenic assays.

Main Results:

• The automated Fiji macro enables high-throughput quantification of nuclear γH2AX fluorescence foci with single-nucleus resolution.
• The standardized workflow ensures reproducibility and cross-sample consistency in DNA double-strand break detection.
• The protocol is versatile, applicable to both nuclear foci and colony-like structures in various assay formats.

Conclusions:

• The presented automated workflow provides a reproducible and standardized method for quantifying DNA damage.
• This protocol facilitates high-throughput analysis of γH2AX foci, minimizing user bias.
• The adaptable nature of the method makes it suitable for routine laboratory use in DNA damage and clonogenic assays.