Abstract
DNA damage that leads to formation of DNA double-strand breaks (DSBs) induces phosphorylation of histone H2AX on Ser-139 at sites flanking the breakage. Immunocytochemical detection of phosphorylated H2AX (denoted as γH2AX) thus provides a marker of DSBs. The method presented in this chapter describes the detection of γH2AX for revealing the presence of DSBs, combined with differential staining of cellular DNA for revealing the cell cycle phase. The detection of γH2AX is based on indirect immunofluorescence using secondary antibody tagged with fluorescein isothiocyanate (FITC) while DNA is counterstained with propidium iodide (PI). Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase.
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This work was supported by NCI grant RO1 28704.
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Huang, X., Darzynkiewicz, Z. (2006). Cytometric Assessment of Histone H2AX Phosphorylation. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 314. Humana Press. https://doi.org/10.1385/1-59259-973-7:073
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DOI: https://doi.org/10.1385/1-59259-973-7:073
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