Protocol

Chromatin Protocols

Volume 523 of the series Methods in Molecular Biology pp 161-168

Date:

Cytometric Analysis of DNA Damage: Phosphorylation of Histone H2AX as a Marker of DNA Double-Strand Breaks (DSBs)

  • Toshiki TanakaAffiliated withDepartment of Surgery and Clinical Science, Division of Chest Surgery, Graduate School of Medicine, Yamaguchi UniversityDepartment of Pathology, Brander Cancer Research Institute, New York Medical College
  • , Dorota HalickaAffiliated withDepartment of Pathology, Brander Cancer Research Institute, New York Medical College
  • , Frank TraganosAffiliated withDepartment of Pathology, Brander Cancer Research Institute, New York Medical College
  • , Zbigniew DarzynkiewiczAffiliated withDepartment of Pathology, Brander Cancer Research Institute, New York Medical College

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Abstract

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 words

γH2AX H2AX phosphorylation DNA double-strand breaks Multiparameter flow cytometry Laser-scanning cytometry Immunocytochemistry Apoptosis