Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells
Summary: 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) microsc...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166721006845 |
| _version_ | 1819099586003730432 |
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| author | Susan Kilgas Anne E. Kiltie Kristijan Ramadan |
| author_facet | Susan Kilgas Anne E. Kiltie Kristijan Ramadan |
| author_sort | Susan Kilgas |
| collection | DOAJ |
| description | Summary: 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). |
| first_indexed | 2024-12-22T00:49:14Z |
| format | Article |
| id | doaj.art-df88efb6bb6f41ffabbd89d98ecc3a22 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-22T00:49:14Z |
| publishDate | 2021-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-df88efb6bb6f41ffabbd89d98ecc3a222022-12-21T18:44:29ZengElsevierSTAR Protocols2666-16672021-12-0124100978Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cellsSusan Kilgas0Anne E. Kiltie1Kristijan Ramadan2MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford OX3 7DQ, UKRowett Institute, University of Aberdeen, Aberdeen AB25 2ZD, UKMRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford OX3 7DQ, UK; Corresponding authorSummary: 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).http://www.sciencedirect.com/science/article/pii/S2666166721006845Cell BiologyCell-based AssaysMicroscopyMolecular BiologyAntibody |
| spellingShingle | Susan Kilgas Anne E. Kiltie Kristijan Ramadan Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells STAR Protocols Cell Biology Cell-based Assays Microscopy Molecular Biology Antibody |
| title | Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells |
| title_full | Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells |
| title_fullStr | Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells |
| title_full_unstemmed | Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells |
| title_short | Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells |
| title_sort | immunofluorescence microscopy based detection of ssdna foci by brdu in mammalian cells |
| topic | Cell Biology Cell-based Assays Microscopy Molecular Biology Antibody |
| url | http://www.sciencedirect.com/science/article/pii/S2666166721006845 |
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