5-Bromo-2'-deoxyuridine (BrdU) labelling and immunostaining is commonly used for the detection of DNA replication using specific antibodies. Previously, we found that these antibodies significantly differ in their affinity to BrdU. Our present data showed that one of the reasons for the differences in the replication signal is the speed of antibody dissociation. Whereas highly efficient antibodies created stable complexes with BrdU, the low efficiency antibodies were unstable. A substantial loss of the signal occurred within several minutes. The increase of the complex stability can be achieved by i) formaldehyde fixation or ii) a quick reaction with a secondary antibody. These steps allowed the same or even higher signal/background ratio to be reached as in the highly efficient antibodies. Based on our findings, we optimised an approach for the fully enzymatic detection of BrdU enabling the fast detection of replicational activity without a significant effect on the tested proteins or the fluorescence of the fluorescent proteins. The method was successfully applied for image and flow cytometry. The speed of the method is comparable to the approach based on 5-ethynyl-2'-deoxyuridine. Moreover, in the case of short labelling pulses, the optimised method is even more sensitive. The approach is also applicable for the detection of 5-trifluoromethyl-2'-deoxyuridine.

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacký University in Olomouc Olomouc Czech Republic

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Ageno M, Dore E, Frontali C. The alkaline denaturation of DNA. Biophysical journal. 1969;9(11): 1281–311. 10.1016/S0006-3495(69)86452-0 PubMed DOI PMC

Dimitrova DS, Berezney R. The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells. Journal of cell science. 2002;115(Pt 21): 4037–51. PubMed

Jackson DA, Pombo A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. The Journal of cell biology. 1998;140(6): 1285–95. PubMed PMC

Kennedy BK, Barbie DA, Classon M, Dyson N, Harlow E. Nuclear organization of DNA replication in primary mammalian cells. Genes & development. 2000;14(22): 2855–68. PubMed PMC

Ligasova A, Strunin D, Liboska R, Rosenberg I, Koberna K. Atomic scissors: a new method of tracking the 5-bromo-2'-deoxyuridine-labeled DNA in situ. PloS one. 2012;7(12): e52584 10.1371/journal.pone.0052584 PubMed DOI PMC

Stanojcic S, Lemaitre JM, Brodolin K, Danis E, Mechali M. In Xenopus egg extracts, DNA replication initiates preferentially at or near asymmetric AT sequences. Mol Cell Biol. 2008;28(17): 5265–74. 10.1128/MCB.00181-08 PubMed DOI PMC

Tang X, Falls DL, Li X, Lane T, Luskin MB. Antigen-retrieval procedure for bromodeoxyuridine immunolabeling with concurrent labeling of nuclear DNA and antigens damaged by HCl pretreatment. J Neurosci. 2007;27(22): 5837–44. 10.1523/JNEUROSCI.5048-06.2007 PubMed DOI PMC

Salic A, Mitchison TJ. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(7): 2415–20. 10.1073/pnas.0712168105 PubMed DOI PMC

Liboska R, Ligasova A, Strunin D, Rosenberg I, Koberna K. Most anti-BrdU antibodies react with 2'-deoxy-5-ethynyluridine—the method for the effective suppression of this cross-reactivity. PloS one. 2012;7(12): e51679 10.1371/journal.pone.0051679 PubMed DOI PMC

Cristofoli WA, Wiebe LI, De Clercq E, Andrei G, Snoeck R, Balzarini J, et al. 5-alkynyl analogs of arabinouridine and 2'-deoxyuridine: cytostatic activity against herpes simplex virus and varicella-zoster thymidine kinase gene-transfected cells. Journal of medicinal chemistry. 2007;50(12): 2851–7. 10.1021/jm0701472 PubMed DOI

Kohlmeier F, Maya-Mendoza A, Jackson DA. EdU induces DNA damage response and cell death in mESC in culture. Chromosome research: an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 2013;21(1): 87–100. PubMed PMC

Ligasova A, Strunin D, Friedecky D, Adam T, Koberna K. A fatal combination: a thymidylate synthase inhibitor with DNA damaging activity. PloS one. 2015;10(2): e0117459 10.1371/journal.pone.0117459 PubMed DOI PMC

Hong V, Steinmetz NF, Manchester M, Finn MG. Labeling live cells by copper-catalyzed alkyne—azide click chemistry. Bioconjug Chem. 2010;21(10): 1912–6. 10.1021/bc100272z PubMed DOI PMC

Loschberger A, Niehorster T, Sauer M. Click chemistry for the conservation of cellular structures and fluorescent proteins: ClickOx. Biotechnol J. 2014;9(5): 693–7. 10.1002/biot.201400026 PubMed DOI

Diermeier-Daucher S, Clarke ST, Hill D, Vollmann-Zwerenz A, Bradford JA, Brockhoff G. Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry A. 2009;75(6): 535–46. 10.1002/cyto.a.20712 PubMed DOI

Ross HH, Rahman M, Levkoff LH, Millette S, Martin-Carreras T, Dunbar EM, et al. Ethynyldeoxyuridine (EdU) suppresses in vitro population expansion and in vivo tumor progression of human glioblastoma cells. J Neurooncol. 2011;105(3): 485–98. 10.1007/s11060-011-0621-6 PubMed DOI PMC

Ligasova A, Liboska R, Rosenberg I, Koberna K. The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions. PloS one. 2015;10(7): e0132393 10.1371/journal.pone.0132393 PubMed DOI PMC

Ligasova A, Raska I, Koberna K. Organization of human replicon: singles or zipping couples? J Struct Biol. 2009;165(3): 204–13. 10.1016/j.jsb.2008.11.004 PubMed DOI PMC

Gonchoroff NJ, Greipp PR, Kyle RA, Katzmann JA. A monoclonal antibody reactive with 5-bromo-2-deoxyuridine that does not require DNA denaturation. Cytometry. 1985;6(6): 506–12. 10.1002/cyto.990060604 PubMed DOI

Takagi S, McFadden ML, Humphreys RE, Woda BA, Sairenji T. Detection of 5-bromo-2-deoxyuridine (BrdUrd) incorporation with monoclonal anti-BrdUrd antibody after deoxyribonuclease treatment. Cytometry. 1993;14(6): 640–8. 10.1002/cyto.990140608 PubMed DOI

Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7(10): R100 10.1186/gb-2006-7-10-r100 PubMed DOI PMC

Kamentsky L, Jones TR, Fraser A, Bray MA, Logan DJ, Madden KL, et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics. 2011;27(8): 1179–80. 10.1093/bioinformatics/btr095 PubMed DOI PMC

Kitao H, Morodomi Y, Niimi S, Kiniwa M, Shigeno K, Matsuoka K, et al. The antibodies against 5-bromo-2'-deoxyuridine specifically recognize trifluridine incorporated into DNA. Sci Rep. 2016;6: 25286 10.1038/srep25286 PubMed DOI PMC

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