Skip to main content
SpringerLink
Log in

Identification of Proliferating and Migrating Cells by BrdU and Other Thymidine Analogs: Benefits and Limitations

  • Protocol
  • First Online:
Immunocytochemistry and Related Techniques

Part of the book series: Neuromethods ((NM,volume 101))

Abstract

Tritiated thymidine ([3H]dT ) and its analogs bromodeoxyuridine (BrdU ), iododeoxyuridine (IdU ), chlorodeoxyuridine (CldU ), and ethynyl deoxyuridine (EdU ) are used extensively to study neuronal time of origin and pattern of migration . Here, we discuss the advantages and pitfalls of identifying dividing cells with these markers and emphasize that they simply indicate DNA synthesis . Thus, in addition to dividing cells, they can label cells that are undergoing DNA repair and/or cell death . As foreign molecules in the DNA, these markers can affect the kinetics of cell proliferation and also have unpredictable functional consequences. We review general protocols and present evidence, based on our own studies in nonhuman primates , that cell proliferation , as indicated by cell numbers and/or their survival , as well as the localization of migrating cells, may be affected by the labeling method itself. We show that measurements made using [3H]dT labeling are more accurate than those using BrdU, perhaps because [3H]dT is less toxic . Thus, utmost caution should be exercised when interpreting the results obtained by using thymidine analogs as possible indicators of the number of mitotic divisions , patterns of migration, final positions , and ultimate fate of cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Duque A, Rakic P (2011) Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate. J Neurosci 31:15205–15217

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Angevine JB Jr (1965) Time of neuron origin in the hippocampal region. An autoradiographic study in the mouse. Exp Neurol S2:1–70

    Google Scholar 

  3. Rakic P (1974) Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition. Science 183:425–427

    Article  CAS  PubMed  Google Scholar 

  4. Rakic P (2002) Adult neurogenesis in mammals: an identity crisis. J Neurosci 22:614–618

    PubMed  Google Scholar 

  5. Rakic P (2002) Neurogenesis in adult primate neocortex: an evaluation of the evidence. Nat Rev Neurosci 3:65–71

    Article  CAS  PubMed  Google Scholar 

  6. Rakic P, Sidman RL (1968) Supravital DNA synthesis in developing human and mouse brain. J Neuropathol Exp Neurol 27:246–276

    Article  CAS  PubMed  Google Scholar 

  7. Schlessinger AR, Cowan WM, Gottlieb DI (1975) An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat. J Comp Neurol 159:149–175

    Article  CAS  PubMed  Google Scholar 

  8. Sidman RL, Miale IL, Feder N (1959) Cell proliferation and migration in the primitive ependymal zone: an autoradiographic study of histogenesis in the nervous system. Exp Neurol 1:322–333

    Article  CAS  PubMed  Google Scholar 

  9. Taupin P (2007) BrdU immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validation. Brain Res Rev 53:198–214

    Article  CAS  PubMed  Google Scholar 

  10. Bisconte JC (1979) Kinetics analysis of cellular populations by means of the quantitative radioautography. Int Rev Cytol 57:75–126

    Article  CAS  PubMed  Google Scholar 

  11. Rogers AW (1973) Techniques of autoradiography. Elsevier, Amsterdam

    Google Scholar 

  12. Sidman RL (1970) Autoradiographic methods and principles for study of the nervous system with thymidine-H3. Springer, New York, NY, pp 252–274

    Google Scholar 

  13. Nowakowski RS, Rakic P (1974) Clearance rate of exogenous 3H-thymidine from the plasma of pregnant rhesus monkeys. Cell Tissue Kinet 7:189–194

    CAS  PubMed  Google Scholar 

  14. Nowakowski RS, Hayes NL (2000) New neurons: extraordinary evidence or extraordinary conclusion? Science 288:771

    Article  CAS  PubMed  Google Scholar 

  15. Magavi SS, Macklis JD (2002) Identification of newborn cells by BrdU labeling and immunocytochemistry in vivo. Meth Mol Biol 198:283–290

    CAS  Google Scholar 

  16. Kornack DR, Rakic P (2001) Cell proliferation without neurogenesis in adult primate neocortex. Science 294:2127–2130

    Article  CAS  PubMed  Google Scholar 

  17. Magavi SS, Leavitt BR, Macklis JD (2000) Induction of neurogenesis in the neocortex of adult mice. Nature 405:951–955

    Article  CAS  PubMed  Google Scholar 

  18. Breunig JJ, Arellano JI, Macklis JD et al (2007) Everything that glitters isn’t gold: a critical review of postnatal neural precursor analyses. Cell Stem Cell 1:612–627

    Article  CAS  PubMed  Google Scholar 

  19. Chehrehasa F, Meedeniya AC, Dwyer P et al (2009) EdU, a new thymidine analogue for labelling proliferating cells in the nervous system. J Neurosci Meth 177:122–130

    Article  CAS  Google Scholar 

  20. Tang X, Falls DL, Li X et al (2007) Antigen-retrieval procedure for bromodeoxyuridine immunolabeling with concurrent labeling of nuclear DNA and antigens damaged by HCl pretreatment. J Neurosci 27:5837–5844

    Article  CAS  PubMed  Google Scholar 

  21. Hammers HJ, Schlenke P (2001) Ultraviolet-induced detection of halogenated pyrimidines (UVID). Curr Protoc Cytom Chapter 7: Unit 7 15

    Google Scholar 

  22. Salic A, Mitchison TJ (2015) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105:2415–2420

    Article  Google Scholar 

  23. Hsu TL, Hanson SR, Kishikawa K et al (2007) Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells. Proc Natl Acad Sci U S A 104:2614–2619

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Sawa M, Hsu TL, Itoh T et al (2006) Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo. Proc Natl Acad Sci U S A 103:12371–12376

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Tornoe CW, Christensen C, Meldal M (2002) Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J Org Chem 67:3057–3064

    Article  CAS  PubMed  Google Scholar 

  26. Maslov AY, Barone TA, Plunkett RJ et al (2004) Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice. J Neurosci 24:1726–1733

    Article  CAS  PubMed  Google Scholar 

  27. Vega CJ, Peterson DA (2005) Stem cell proliferative history in tissue revealed by temporal halogenated thymidine analog discrimination. Nat Methods 2:167–169

    Article  CAS  PubMed  Google Scholar 

  28. Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435:406–417

    Article  CAS  PubMed  Google Scholar 

  29. Bannigan J, Langman J (1979) The cellular effect of 5-bromodeoxyuridine on the mammalian embryo. J Embryol Exp Morphol 50:123–135

    CAS  PubMed  Google Scholar 

  30. Webster W, Shimada M, Langman J (1973) Effect of fluorodeoxyuridine, colcemid, and bromodeoxyuridine on developing neocortex of the mouse. Am J Anat 137:67–85

    Article  CAS  PubMed  Google Scholar 

  31. Kornack DR, Rakic P (1998) Changes in cell-cycle kinetics during the development and evolution of primate neocortex. Proc Natl Acad Sci U S A 95:1242–1246

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Leuner B, Glasper ER, Gould E (2009) Thymidine analog methods for studies of adult neurogenesis are not equally sensitive. J Comp Neurol 517:123–133

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Shibui S, Hoshino T, Vanderlaan M et al (1989) Double labeling with iodo- and bromodeoxyuridine for cell kinetics studies. J Histochem Cytochem 37:1007–1011

    Article  CAS  PubMed  Google Scholar 

  34. Gratzner HG (1982) Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 218:474–475

    Article  CAS  PubMed  Google Scholar 

  35. Imamura F, Ayoub AE, Rakic P et al (2011) Timing of neurogenesis is a determinant of olfactory circuitry. Nat Neurosci 14:331–337

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. del Rio JA, Soriano E (1989) Immunocytochemical detection of 5′-bromodeoxyuridine incorporation in the central nervous system of the mouse. Brain Res Dev Brain Res 49:311–317

    Article  PubMed  Google Scholar 

  37. Miller MW, Nowakowski RS (1988) Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res 457:44–52

    Article  CAS  PubMed  Google Scholar 

  38. Ehmann UK, Williams JR, Nagle WA et al (1975) Perturbations in cell cycle progression from radioactive DNA precursors. Nature 258:633–636

    Article  CAS  PubMed  Google Scholar 

  39. Kolb B, Pedersen B, Ballermann M et al (1999) Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities. J Neurosci 19:2337–2346

    CAS  PubMed  Google Scholar 

  40. Kuwagata M, Ogawa T, Nagata T et al (2007) The evaluation of early embryonic neurogenesis after exposure to the genotoxic agent 5-bromo-2′-deoxyuridine in mice. Neurotoxicology 28:780–789

    Article  CAS  PubMed  Google Scholar 

  41. Sekerkova G, Ilijic E, Mugnaini E (2004) Bromodeoxyuridine administered during neurogenesis of the projection neurons causes cerebellar defects in rat. J Comp Neurol 470:221–239

    Article  PubMed  Google Scholar 

  42. Nowakowski RS, Lewin SB, Miller MW (1989) Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population. J Neurocytol 18:311–318

    Article  CAS  PubMed  Google Scholar 

  43. Burns KA, Ayoub AE, Breunig JJ et al (2007) Nestin-CreER mice reveal DNA synthesis by nonapoptotic neurons following cerebral ischemia hypoxia. Cereb Cortex 17:2585–2592

    Article  PubMed  Google Scholar 

  44. Kuan CY, Schloemer AJ, Lu AG et al (2004) Hypoxia-ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain. J Neurosci 24:10763–10772

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Yang Y, Geldmacher DS, Herrup K (2001) DNA replication precedes neuronal cell death in Alzheimer’s disease. J Neurosci 21:2661–2668

    CAS  PubMed  Google Scholar 

  46. Brockman RW, Anderson EP (1963) Biochemistry of cancer (metabolic aspects). Annu Rev Biochem 32:463–512

    Article  CAS  PubMed  Google Scholar 

  47. Hitchings G, Elion G (1967) Mechanisms of action of purine and pyrimidine analogs. In: Brodsky I, Kahn S, Moyer J et al (eds) Cancer chemotherapy I. Grune and Stratton, New York, NY, p 26

    Google Scholar 

  48. Roy-Burman P (1970) Analogues of nucleic acid components. Springer, New York, NY

    Book  Google Scholar 

  49. Rash BG, Lim HD, Breunig JJ, Vaccarino FM (2011) FGF signaling expands embryonic cortical surface area by regulating Notch-dependent neurogenesis. J Neurosci 31:15604–15617

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Drs. Albert Ayoub and Brian Rash for providing images and for useful discussion on the protocols used in their research. We also thank Ms. Mariamma Pappy for useful discussion. This work was supported by a grant from NIH NINDS.

Author information

Authors and Affiliations

  1. Department of Neurobiology, Kavli Institute for Neuroscience, Yale University School of Medicine, 333 Cedar Street, 208001, New Haven, CT, 06520, USA

    Alvaro Duque & Pasko Rakic

Authors
  1. Alvaro Duque
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pasko Rakic
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pasko Rakic .

Editor information

Editors and Affiliations

  1. Department of Veterinary Sciences, University of Torino, Grugliasco, Torino, Torino, Italy

    Adalberto Merighi

  2. Department of Veterinary Sciences, University of Torino, Grugliasco, Torino, Torino, Italy

    Laura Lossi

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Duque, A., Rakic, P. (2015). Identification of Proliferating and Migrating Cells by BrdU and Other Thymidine Analogs: Benefits and Limitations. In: Merighi, A., Lossi, L. (eds) Immunocytochemistry and Related Techniques. Neuromethods, vol 101. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2313-7_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2313-7_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2312-0

  • Online ISBN: 978-1-4939-2313-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation