Cytometric Analysis of DNA Damage: Phosphorylation of Histone H2AX as a Marker of DNA Double-Strand Breaks (DSBs)
Phosphorylation of histone H2AX on Ser 139 is a sensitive reporter of DNA damage, particularly if the damage involves induction of DNA double-strand breaks (DSBs). Phosphorylated H2AX has been named γH2AX and its presence in the nucleus can be detected immunocytochemically. Multiparameter analysis of γH2AX immunofluorescence by flow or laser-scanning cytometry allows one to measure extent of DNA damage in individual cells and to correlate it with their position in the cell cycle and induction of apoptosis. This chapter presents the protocols and outlines applications of multiparameter cytometry in analysis of H2AX phosphorylation as a reporter of the presence of DSBs.
Key wordsγH2AX H2AX phosphorylation DNA double-strand breaks Multiparameter flow cytometry Laser-scanning cytometry Immunocytochemistry Apoptosis
- 8.Furuta T, Takemura H, Liao-Z-Y, Aune GJ, Redon C, Sedelnikova OA, Pilch DR, Rogakou EP, Celeste A, Chen HT, Nussenzweig A, Aladjem MI, Bonner WM, Pommier Y. (2003) Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induces by mammalian topoisomerase I cleavage complexes. J Biol Chem. 278, 20303–20312.PubMedCrossRefGoogle Scholar
- 22.Huang X, Kurose A, Tanaka T, Traganos F, Dai W, Darzynkiewicz Z (2006) Sequential phosphorylation of Ser-10 on histone H3 and Ser-139 on histone H2AX and ATM activation during premature chromosome condensation: Relationship to cell-cycle and apoptosis. Cytometry A 69A, 222–229.CrossRefGoogle Scholar