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...

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Published in:STAR protocols Vol. 2; no. 4; p. 100978
Main Authors: Kilgas, Susan, Kiltie, Anne E., Ramadan, Kristijan
Format: Journal Article
Language:English
Published: United States Elsevier Inc 17.12.2021
Elsevier
Subjects:
Antibody
Bromodeoxyuridine - chemistry
Bromodeoxyuridine - metabolism
Cell Biology
Cell Line, Tumor
Cell-based Assays
DNA, Single-Stranded - analysis
DNA, Single-Stranded - chemistry
DNA, Single-Stranded - genetics
DNA, Single-Stranded - metabolism
Genomic Instability - genetics
Humans
Microscopy
Microscopy, Fluorescence - methods
Molecular Biology
Protocol
Microscopy
Molecular Biology
Antibody
Cell-based Assays
Cell Biology
ISSN:2666-1667, 2666-1667
Online Access:Get full text
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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). [Display omitted] •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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ISSN:2666-1667
2666-1667
DOI:10.1016/j.xpro.2021.100978