Huang Xuan, Halicka H Dorota, Darzynkiewicz Zbigniew
Brander Cancer Research Institute, Valhalla, New York, USA.
Curr Protoc Cytom. 2004 Nov;Chapter 7:Unit 7.27. doi: 10.1002/0471142956.cy0727s30.
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.
本单元介绍了用于揭示DNA双链断裂存在的磷酸化组蛋白H2AX的免疫细胞化学检测方法。双链断裂表明由电离辐射或用抗肿瘤药物（如DNA拓扑异构酶抑制剂）处理诱导的DNA损伤。然而，双链断裂也可能是内在的，由于各种原因在健康的未处理细胞中发生，并在凋亡细胞的DNA片段化过程中产生。本单元介绍了区分辐射或药物诱导的断裂与未处理细胞中内在形成的或与凋亡相关的断裂的策略。该方案描述了对Ser-139位点磷酸化的组蛋白H2AX进行免疫细胞化学检测，并结合DNA含量测量，以识别具有DNA双链断裂的细胞，并同时评估其细胞周期阶段。检测基于使用异硫氰酸荧光素（FITC）标记的二抗的间接免疫荧光，DNA用碘化丙啶（PI）复染。可能被PI染色的细胞RNA用核糖核酸酶A去除。
