Unit

UNIT 7.27 Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage (DNA Double-Strand Breaks)

  1. Xuan Huang,
  2. H. Dorota Halicka,
  3. Zbigniew Darzynkiewicz

Published Online: 1 NOV 2004

DOI: 10.1002/0471142956.cy0727s30

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Huang, X., Halicka, H. D. and Darzynkiewicz, Z. 2004. Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage (DNA Double-Strand Breaks). Current Protocols in Cytometry. 30:7.27.1–7.27.7.

Author Information

  1. Brander Cancer Research Institute, New York Medical College, Valhalla, New York

Publication History

  1. Published Online: 1 NOV 2004
  2. Published Print: OCT 2004

Abstract

This unit describes immunocytochemical detection of phosphorylated histone H2AX for revealing the presence of DNA double-strand breaks. Double-strand breaks indicate DNA damage induced by ionizing radiation or by treatment with antitumor drugs such as DNA topoisomerase inhibitors. However, double-strand breaks can also be intrinsic, occurring in healthy, nontreated cells for a variety of reasons, and are generated in the course of DNA fragmentation in apoptotic cells. The unit presents strategies to distinguish radiation- or drug-induced breaks from those intrinsically formed in untreated cells or associated with apoptosis. The protocol describes the immunocytochemical detection of histone H2AX phosphorylated on Ser-139 combined with measurement of DNA content to identify cells that have DNA double-strand breaks and to concurrently assess their cell cycle phase. The detection is based on indirect immunofluorescence using a FITC-labeled secondary antibody, and DNA is counterstained with propidium iodide (PI). Cellular RNA, which may be stained by PI, is removed with RNase A.

Keywords:

  • DNA damage;
  • double-strand breaks;
  • phosphorylated histone;
  • flow cytometry;
  • immunofluorescence