title: Detection of histone H2AX phosphorylation on Ser-139 as an indicator of DNA damage (DNA double-strand breaks).
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.
authors.affiliation: Brander Cancer Research Institute, Valhalla, New York, USA.
authors.forename: Xuan
authors.initials: X
authors.lastname: Huang
authors.forename: H Dorota
authors.initials: HD
authors.lastname: Halicka
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
mesh_terms: D002453:Cell Cycle; D004247:DNA / Q000032:analysis; D053903:DNA Breaks, Double-Stranded; D006657:Histones / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D010766:Phosphorylation; D011419:Propidium; D012694:Serine / Q000378:metabolism
chemical_list: C468783:H2AX protein, human; D006657:Histones; D011419:Propidium; D012694:Serine; D004247:DNA