Computational analysis of the number,area and density of γ-H2AX foci in breast cancer cells exposed to 111In-DTPA-hEGF or γ-rays using Image-J software

Purpose: To develop a simple method for the quantification of γ-H2AX focus number, density and size.

Methods: MDA-MB-468 human breast cancer cells were treated overnight with ¹¹¹In-diethylenetriaminepentaacetic acid human epidermal growth factor (¹¹¹In-DTPA-hEGF, 0–142 kBq/pmol) or exposed to γ-radiation to induce DNA double strand breaks (DSB). DNA DSB formation was evaluated by detection of phosphorylated histone H2AX on serine 139 (γ-H2AX) using immunofluorescence. Confocal microscopy was used to capture images of γ-H2AX foci and cell nuclei. Image-J software with customized macros was used to quantify γ-H2AX foci.

Results: The number of γ-H2AX foci per nucleus scored using Image-J correlated strongly with the number scored using direct visual confirmation (coefficient of determination, R² = 0.950; 60 nuclei scored). The mean density (grayscale values per pixel), area and integrated density (IntDen) of individual foci increased linearly as the specific radioactivity (SR) increased up to 67 kBq/pmol (R² values of 0.826, 0.964, 0.978, respectively). The mean number of foci per nucleus, the combined area of γ-H2AX foci per nucleus and the IntDen per nucleus also increased linearly with SR, giving R² values of 0.926, 0.974 and 0.983, respectively. Similar linear relationships were observed with the γ-ray absorbed dose up to 3.0 Gy.

Conclusions: The density, area and IntDen of individual foci, as well as the number of γ-H2AX foci, total focus area and IntDen per nucleus were successfully quantified using Image-J with customized macros.