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

Xuan Huang1, H. Dorota Halicka1, Zbigniew Darzynkiewicz1

1 Brander Cancer Research Institute, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.27
DOI:  10.1002/0471142956.cy0727s30
Online Posting Date:  November, 2004
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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

     
 
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Table of Contents

  • Basic Protocol 1: Detection of H2AX Phosphorylated on SER‐139 in Relation to the Cell Cycle Phase
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of H2AX Phosphorylated on SER‐139 in Relation to the Cell Cycle Phase

  Materials
  • Cells
  • Medium
  • Agents expected to induce DSBs
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1% methanol‐free formaldehyde (Polysciences) in PBS, 4°C (store ≤2 weeks at 4°C)
  • 70% ethanol, 0° to 4°C
  • 1% (w/v) BSA and 0.2% (v/v) Triton X‐100 in PBS (BSA‐T‐PBS; store ≤2 weeks at 4°C)
  • Unconjugated primary γH2AX antibody: murine monoclonal anti–histone γH2AX antibody (Upstate Biotechnology) or rabbit polyclonal anti–histone γH2AX antibody (Trevigen)
  • FITC‐conjugated secondary antibody, appropriately titered: e.g., polyclonal goat anti‐mouse or anti‐rabbit‐F(ab′)2, depending on the source of the primary antibody
  • Propidium iodide (PI) staining solution: 5 µl/ml PI (Molecular Probes) and 100 µl/ml DNase‐free RNase A (Sigma) in PBS (store ≤2 weeks protected from light at 4°C)
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer with blue‐light (e.g., 488‐nm argon‐ion laser or BG‐12 excitation filter) excitation source and filters for collection of green (530 ± 20 nm) and red (>600 nm) fluorescence
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Figures

Videos

Literature Cited

Literature Cited
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