Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells
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...
| Main Authors: | , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
Cell Press
2021
|
| _version_ | 1797106561699545088 |
|---|---|
| author | Kilgas, S Kiltie, AE Ramadan, K |
| author_facet | Kilgas, S Kiltie, AE Ramadan, K |
| author_sort | Kilgas, S |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T07:04:15Z |
| format | Journal article |
| id | oxford-uuid:1945ae29-f850-4a0f-9354-4c40398ba629 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:04:15Z |
| publishDate | 2021 |
| publisher | Cell Press |
| record_format | dspace |
| spelling | oxford-uuid:1945ae29-f850-4a0f-9354-4c40398ba6292022-04-27T13:29:13ZImmunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cellsJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:1945ae29-f850-4a0f-9354-4c40398ba629EnglishSymplectic ElementsCell Press2021Kilgas, SKiltie, AERamadan, KDNA 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. |
| spellingShingle | Kilgas, S Kiltie, AE Ramadan, K Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells |
| 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 |
| work_keys_str_mv | AT kilgass immunofluorescencemicroscopybaseddetectionofssdnafocibybrduinmammaliancells AT kiltieae immunofluorescencemicroscopybaseddetectionofssdnafocibybrduinmammaliancells AT ramadank immunofluorescencemicroscopybaseddetectionofssdnafocibybrduinmammaliancells |