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

Item request has been placed! ×
Item request cannot be made. ×
loading   Processing Request
Read More Add to Saved list
  • Author(s): Kilgas S;Kilgas S; Kiltie AE; Kiltie AE; Ramadan K; Ramadan K
  • Source:
    STAR protocols [STAR Protoc] 2021 Nov 25; Vol. 2 (4), pp. 100978. Date of Electronic Publication: 2021 Nov 25 (Print Publication: 2021).
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Cell Press Country of Publication: United States NLM ID: 101769501 Publication Model: eCollection Cited Medium: Internet ISSN: 2666-1667 (Electronic) Linking ISSN: 26661667 NLM ISO Abbreviation: STAR Protoc
    • Publication Information:
      Original Publication: [Cambridge, MA] : Cell Press, [2020]-
    • Subject Terms:
      DNA, Single-Stranded*/analysis ; DNA, Single-Stranded*/chemistry ; DNA, Single-Stranded*/genetics ; DNA, Single-Stranded*/metabolism; Bromodeoxyuridine/*chemistry ; Microscopy, Fluorescence/*methods; Bromodeoxyuridine/metabolism ; Cell Line, Tumor ; Genomic Instability/genetics ; Humans
    • 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).
      Competing Interests: The authors declare no competing interests.
      (© 2021 The Authors.)
    • References:
      Oncogene. 2001 Jun 14;20(27):3486-96. (PMID: 11429695)
      J Cell Biol. 2013 Aug 5;202(3):579-95. (PMID: 23897892)
      Cell. 2019 Jan 24;176(3):505-519.e22. (PMID: 30612738)
      Nat Protoc. 2015 Sep;10(9):1297-307. (PMID: 26226461)
      Genes (Basel). 2020 Apr 09;11(4):. (PMID: 32283785)
      Cell. 2013 Nov 21;155(5):1088-103. (PMID: 24267891)
      Mol Cell. 2019 Sep 19;75(6):1299-1314.e6. (PMID: 31353207)
      Cell Rep. 2021 May 25;35(8):109153. (PMID: 34038735)
      Science. 2002 Jul 26;297(5581):599-602. (PMID: 12142537)
      Nat Commun. 2019 Jul 17;10(1):3142. (PMID: 31316063)
      Methods Mol Biol. 2015;1292:67-75. (PMID: 25804748)
      Bio Protoc. 2020 Aug 05;10(15):e3701. (PMID: 33659366)
      Hum Mol Genet. 2010 May 1;19(9):1690-701. (PMID: 20123862)
      Nucleic Acids Res. 2015 Sep 30;43(17):8352-67. (PMID: 26240375)
      Exp Mol Med. 2020 Oct;52(10):1705-1714. (PMID: 33122806)
      Nat Cell Biol. 2016 Mar;18(3):271-280. (PMID: 26807646)
      Methods Mol Biol. 2021;2153:59-69. (PMID: 32840772)
      Nat Commun. 2019 Feb 26;10(1):944. (PMID: 30808869)
      Nat Cell Biol. 2006 Feb;8(2):148-55. (PMID: 16429127)
      Nature. 2018 Nov;563(7732):522-526. (PMID: 30464262)
    • Grant Information:
      23279 United Kingdom CRUK_ Cancer Research UK; MC_PC_12001/1 United Kingdom MRC_ Medical Research Council; MC_UU_00001/1 United Kingdom MRC_ Medical Research Council
    • Contributed Indexing:
      Keywords: Antibody; Cell Biology; Cell-based Assays; Microscopy; Molecular Biology
    • Accession Number:
      0 (DNA, Single-Stranded)
      G34N38R2N1 (Bromodeoxyuridine)
    • Publication Date:
      Date Created: 20211210 Date Completed: 20220405 Latest Revision: 20240210
    • Publication Date:
      20240210
    • Accession Number:
      PMC8634038
    • Accession Number:
      10.1016/j.xpro.2021.100978
    • Accession Number:
      34888531