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

Purpose: To develop a simple method for the quantification of -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, 0-142kBq/pmol) or exposed to -radiation to induce DNA double strand breaks (DSB). DNA DSB formation was evaluated by detection of phosphorylated histone H2AX on serine 139 (-H2AX) using immunofluorescence. Confocal microscopy was used to capture images of -H2AX foci and cell nuclei. Image-J software with customized macros was used to quantify -H2AX foci. Results: The number of -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 67kBq/pmol (R2 values of 0.826, 0.964, 0.978, respectively). The mean number of foci per nucleus, the combined area of -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 -ray absorbed dose up to 3.0Gy. Conclusions: The density, area and IntDen of individual foci, as well as the number of -H2AX foci, total focus area and IntDen per nucleus were successfully quantified using Image-J with customized macros.