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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Vydáno v:	STAR protocols Ročník 2; číslo 4; s. 100978
Hlavní autoři:	Kilgas, Susan, Kiltie, Anne E., Ramadan, Kristijan
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	United States Elsevier Inc 17.12.2021 Elsevier
Témata:	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
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Shrnutí:	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.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Technical contact Lead contact
ISSN:	2666-1667 2666-1667
DOI:	10.1016/j.xpro.2021.100978