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

Susan Kilgas; Anne E. Kiltie; Kristijan Ramadan

doi:10.1016/j.xpro.2021.100978

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

Susan Kilgas, Anne E. Kiltie, Kristijan Ramadan^*

^*Corresponding author for this work

The Rowett Institute of Nutrition and Health

Research output: Contribution to journal › Article › peer-review

Abstract

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).

Original language	English
Article number	100978
Number of pages	15
Journal	STAR Protocols
Volume	2
Issue number	4
Early online date	25 Nov 2021
DOIs	https://doi.org/10.1016/j.xpro.2021.100978
Publication status	Published - 17 Dec 2021

Keywords

Antibody
Cell Biology
Cell-based Assays
Microscopy
Molecular Biology

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title = "Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells",

abstract = "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).",

keywords = "Antibody, Cell Biology, Cell-based Assays, Microscopy, Molecular Biology",

author = "Susan Kilgas and Kiltie, {Anne E.} and Kristijan Ramadan",

note = "Acknowledgments This work was funded by the MRC Program grant MC_PC 12001/1 and MC_UU_00001/1 to K.R., and S.K. was supported by the MRC Oxford Institute of Radiation Oncology (OIRO) Cancer Research UK (CRUK) Studentship. We thank Dr. Rhodri Wilson from the microscopy imaging core (University of Oxford, Department of Oncology) for his technical advice and assistance. Graphical abstract was created with BioRender.",

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doi = "10.1016/j.xpro.2021.100978",

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AU - Kilgas, Susan

AU - Kiltie, Anne E.

AU - Ramadan, Kristijan

N1 - Acknowledgments This work was funded by the MRC Program grant MC_PC 12001/1 and MC_UU_00001/1 to K.R., and S.K. was supported by the MRC Oxford Institute of Radiation Oncology (OIRO) Cancer Research UK (CRUK) Studentship. We thank Dr. Rhodri Wilson from the microscopy imaging core (University of Oxford, Department of Oncology) for his technical advice and assistance. Graphical abstract was created with BioRender.

PY - 2021/12/17

Y1 - 2021/12/17

N2 - 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).

AB - 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).

KW - Antibody

KW - Cell Biology

KW - Cell-based Assays

KW - Microscopy

KW - Molecular Biology

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DO - 10.1016/j.xpro.2021.100978

M3 - Article

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JO - STAR Protocols

JF - STAR Protocols

SN - 2666-1667

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M1 - 100978

ER -

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

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