期刊
INTERNATIONAL JOURNAL OF RADIATION BIOLOGY
卷 85, 期 3, 页码 262-271出版社
TAYLOR & FRANCIS LTD
DOI: 10.1080/09553000902748757
关键词
-H2AX foci; Image-J; DNA double strand breaks; breast cancer cells; 111In-DTPA-hEGF; -rays
资金
- Canadian Breast Cancer Foundation
- Canadian Breast Cancer Research Alliance
- Canadian Institutes for Health Research Strategic Training Fellow
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.
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