Djerir Billel, Maréchal Alexandre
Faculty of Sciences, Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada.
Methods Mol Biol. 2025;2919:83-89. doi: 10.1007/978-1-0716-4486-7_5.
Cells are constantly exposed to a range of intrinsic and environmental factors, such as reactive oxygen species, ionizing radiation, and chemical agents, which threaten DNA integrity and can induce double-strand breaks (DSBs). DSBs are highly cytotoxic and must be rapidly repaired to maintain genome stability. One of the earliest chromatin marks associated with DSBs is the phosphorylation of histone variant H2A.X on serine 139, which leads to the formation of γ-H2A.X foci detectable by immunofluorescence. Immunocytochemical detection of γ-H2A.X is a widely used method to indirectly assess DSB formation and repair. In this chapter, we describe a simple and efficient immunofluorescence (IF) procedure for detecting DSB formation in U-2 OS cells treated with genotoxic agents. This technique does not require an expensive laboratory setup and can also be used to detect the formation of foci of additional repair factors using appropriate labeling.
细胞不断受到一系列内在和环境因素的影响，如活性氧、电离辐射和化学试剂，这些因素会威胁DNA完整性并可诱导双链断裂（DSB）。DSB具有高度细胞毒性，必须迅速修复以维持基因组稳定性。与DSB相关的最早染色质标记之一是组蛋白变体H2A.X的丝氨酸139位点磷酸化，这会导致通过免疫荧光可检测到的γ-H2A.X焦点的形成。γ-H2A.X的免疫细胞化学检测是一种广泛用于间接评估DSB形成和修复的方法。在本章中，我们描述了一种简单有效的免疫荧光（IF）程序，用于检测用遗传毒性剂处理的U-2 OS细胞中的DSB形成。该技术不需要昂贵的实验室设置，也可用于使用适当的标记检测其他修复因子焦点的形成。
