-H2AX Detection in Somatic and Germ Cells of Mice

The phosphorylation of histone H2AX at serine 139 (γ-H2AX) is one of the first steps of DNA damage response and its detection is widely used as a sensitive marker for DNA double-strand breaks induced by ionizing radiation or other genotoxic agents. Immuno-stained phosphorylated histone can be measured in single cells by flow cytometry or single γ-H2AX foci can be visualized and counted microscopically in histological or cytological preparations. Animal studies are well recognized as important tools to study mechanisms of in vivo response to genotoxic stress. Tissues are composed by many cell types differing for function, differentiation, and proliferative capacity. In particular, due to the complexity of spermatogenesis and the heterogeneity of testicular cell subpopulations, an accurate characterization of damage in this tissue is difficult and requires an approach which allows the identification of damage in the different cellular compartments. This chapter presents techniques for γ-H2AX detection in mouse bone marrow and testicular cells. Furthermore, advantages and weaknesses of flow cytometric and microscopic methods are described.