γ-H2AX Detection in Peripheral Blood Lymphocytes, Splenocytes, Bone Marrow, Xenografts, and Skin
Measurement of DNA double-strand break (DSB) levels in cells is useful in many research areas, including those related to DNA damage and repair, tumorigenesis, anti-cancer drug development, apoptosis, radiobiology, environmental effects, and aging, as well as in the clinic. DSBs can be detected in the nuclei of cultured cells and tissues with an antibody to H2AX phosphorylated on serine residue 139 (γ-H2AX). DSB levels can be obtained either by measuring overall γ-H2AX protein levels in a cell population or by counting γ-H2AX foci in individual nuclei. Total levels can be obtained in extracts of cell populations by immunoblot analysis, and in cell populations by flow cytometry. Furthermore, with flow cytometry, the cell cycle distribution of a population can be obtained in addition to DSB levels, which is an advantage when studying anti-cancer drugs targeting replicating tumor cells. These described methods are used in genotoxicity assays of compounds of interest or in analyzing DSB repair after exposure to drugs or radiation. Immunocyto/immunohistochemical analysis can detect γ-H2AX foci in individual cells and is very sensitive (a single DSB can be visualized), permitting the use of extremely small samples. Measurements of γ-H2AX focal numbers can reveal subtle changes found in the radiation-induced tissue bystander response, low dose radiation exposure, and in cells with mutations in genomic stability maintenance pathways. In addition, marking DNA DSBs in a nucleus with γ-H2AX is a powerful tool to identify novel DNA repair proteins by their abilities to co-localize with γ-H2AX foci at the DSB site. This chapter presents techniques for γ-H2AX detection in a variety of human and mouse samples.
Key wordsγ-H2AX DNA damage Immunofluorescence Immunoblotting Flow cytometry Lymphocytes Splenocytes Bone marrow Xenografts Skin
This work was funded by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research, NIH. B.T., S.L., and R.K. were funded by NCI Contract N01-CO-12400. Human blood samples were obtained from paid healthy volunteers who gave written informed consent to participate in an IRB-approved study for the collection of blood samples for in vitro research use. The protocol is designed to protect subjects from research risks as defined in 45CFR46 and to abide by all internal NIH guidelines for human subjects research (protocol number 99-CC-0168). NCI-Frederick is accredited by AAALAC Interna‑tional and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” (National Research Council, 1996; National Academy Press; Washington, DC). All studies were conducted according to an approved animal care and use committee protocol.
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