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...

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Main Authors:	Susan Kilgas, Anne E. Kiltie, Kristijan Ramadan
Format:	article
Language:	EN
Published:	Elsevier 2021
Subjects:	Cell Biology Cell-based Assays Microscopy Molecular Biology Antibody Science (General) Q1-390
Online Access:	https://doaj.org/article/df88efb6bb6f41ffabbd89d98ecc3a22
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spelling	oai:doaj.org-article:df88efb6bb6f41ffabbd89d98ecc3a222021-11-28T04:38:21ZImmunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells2666-166710.1016/j.xpro.2021.100978https://doaj.org/article/df88efb6bb6f41ffabbd89d98ecc3a222021-12-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2666166721006845https://doaj.org/toc/2666-1667Summary: 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).Susan KilgasAnne E. KiltieKristijan RamadanElsevierarticleCell BiologyCell-based AssaysMicroscopyMolecular BiologyAntibodyScience (General)Q1-390ENSTAR Protocols, Vol 2, Iss 4, Pp 100978- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cell Biology Cell-based Assays Microscopy Molecular Biology Antibody Science (General) Q1-390
spellingShingle	Cell Biology Cell-based Assays Microscopy Molecular Biology Antibody Science (General) Q1-390 Susan Kilgas Anne E. Kiltie Kristijan Ramadan Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells
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).
format	article
author	Susan Kilgas Anne E. Kiltie Kristijan Ramadan
author_facet	Susan Kilgas Anne E. Kiltie Kristijan Ramadan
author_sort	Susan Kilgas
title	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_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_sort	immunofluorescence microscopy-based detection of ssdna foci by brdu in mammalian cells
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/df88efb6bb6f41ffabbd89d98ecc3a22
work_keys_str_mv	AT susankilgas immunofluorescencemicroscopybaseddetectionofssdnafocibybrduinmammaliancells AT anneekiltie immunofluorescencemicroscopybaseddetectionofssdnafocibybrduinmammaliancells AT kristijanramadan immunofluorescencemicroscopybaseddetectionofssdnafocibybrduinmammaliancells
_version_	1718408289368145920

Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells

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