Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells.
Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells.
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DOI：
10.1016/j.xpro.2021.100978
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发表时间：
2021-12-17
期刊：
影响因子：
--
通讯作者：
Ramadan K
中科院分区：
文献类型：
--
作者：
Kilgas S;Kiltie AE;Ramadan K
DNA末端切除将双链DNA （dsDNA）的断端转化为3 ' -单链DNA （3 ' -ssDNA）。切除的程度调节DNA双链断裂（DSB）修复途径的选择，从而调节基因组的稳定性。在这里，我们描述了一种优化的基于免疫荧光（IF）显微镜的方案，通过用5-溴-2 ' -脱氧尿苷（BrdU）标记基因组DNA来测量哺乳动物细胞中的ssDNA。抗BrdU抗体可以在非变性条件下检测到BrdU灶，为大多数哺乳动物细胞系的DNA末端切除提供了准确可靠的读数。有关该协议使用和执行的完整细节，请参阅Kilgas等人（2021）。非变性条件下BrdU标记单链DNA （ssDNA）检测基于免疫荧光（IF）共聚焦显微镜测量的ssDNA焦点数量与细胞周期标记物共染色兼容可用于定量的半定量图像分析DNA末端切除将双链DNA （dsDNA）的断端转化为3 ' -单链DNA （3 ' -ssDNA）。切除的程度调节DNA双链断裂（DSB）修复途径的选择，从而调节基因组的稳定性。在这里，我们描述了一种优化的基于免疫荧光（IF）显微镜的方案，通过用5-溴-2 ' -脱氧尿苷（BrdU）标记基因组DNA来测量哺乳动物细胞中的ssDNA。抗BrdU抗体可以在非变性条件下检测到BrdU灶，为大多数哺乳动物细胞系的DNA末端切除提供了准确可靠的读数。
DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3′-single-stranded DNA (3′-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2′-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines. For complete details on the use and execution of this protocol, please refer to Kilgas et al. (2021). Single-stranded DNA (ssDNA) detection by BrdU labeling under non-denaturing conditions Number of ssDNA foci measured by immunofluorescence (IF)-based confocal microscopy Compatible with co-staining for cell cycle markers Amenable to semi-quantitative image analysis for quantification DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3′-single-stranded DNA (3′-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2′-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines.
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