Development of an Automated γ-H2AX Immunocytochemistry Assay

Abstract Hou, Y-N., Lavaf, A., Huang, D., Peters, S., Huq, R., Friedrich, V., Rosenstein, B. S. and Kao, J. Development of an Automated γ-H2AX Immunocytochemistry Assay. Radiat. Res. 171, 360–367 (2009). γ-H2AX is emerging as an important marker of ionizing radiation-induced double-strand breaks. Development of a significantly automated method to quantify γ-H2AX would have broad application in assessing physiological responses to radiation exposure. PC-3 and DU145 prostate cancer cells grown on glass cover slips and 96-well plates were irradiated and assessed for γ-H2AX focus formation by immunofluorescence analysis. The γ-H2AX immunofluorescence staining was performed either manually or by using a preprogrammed automated robotic liquid handling system. A computer-controlled charge-coupled device camera acquired images serially throughout the thickness of each cell. Image analysis was performed manually and/or with automated image segmentation software. A robust relationship between radiation dose and γ-H2AX focus numbers was demonstrated with both manual and automated image analysis methods, with excellent agreement observed between the two techniques. The r2 correlation coefficients and Z factors exceeded 0.9 and 0.5, respectively, when γ-H2AX focus formation was correlated with radiation dose using the automated technique. Inhibition of γ-H2AX foci by drugs readily detected with this assay. Robotic specimen preparation with automated image acquisition and analysis can be used to quantify γ-H2AX foci in irradiated cells, and the results agree well those obtained by manual counts. These data suggest that this assay has an excellent signal-to-noise ratio and is suitable for high-throughput applications.

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