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Journal of Molecular Histology

, Volume 49, Issue 3, pp 229–234 | Cite as

BrdU/EdU dual labeling to determine the cell-cycle dynamics of defined cellular subpopulations

Short Communication

Abstract

Measuring the mean duration of synthesis-phase (Ts) and of the total cell-cycle (Tc) within progenitor cell populations can provide important insights into the biology governing these cells. Rather than a passive process that shows little variability across cellular contexts, the cell-cycle is instead highly regulated. For example, in the rodent forebrain, Ts is selectively lengthened in radial glial progenitor cells undergoing symmetric versus asymmetric division. This lengthening is thought to minimize the potential for copying errors that can occur during DNA replication. Manipulating cell-cycle duration can also affect cell fate, demonstrating that in certain circumstances cell-cycle duration is an instructive process. Currently, cell-cycle length is typically measured using either cumulative labeling with a single thymidine analogue, or via dual thymidine analogue labeling approaches. However, these methods are often time-consuming and inefficient. Here, using the embryonic mouse cerebral cortex as a model system, we describe a simplified dual thymidine analogue protocol using BrdU and EdU that can be used to measure Ts and Tc. The advantage of this protocol over cumulative labeling approaches is that only a single time-point is required for measurement. An additional benefit of this protocol over existing dual-analog approaches (CldU/IdU) is the antibody-free detection of EdU and the acid-free detection of BrdU, processes allowing for the parallel use of specific antibodies so as to measure the cell-cycle in immunologically defined cellular subpopulations.

Keywords

Cell-cycle Dual labeling Cumulative labeling BrdU EdU S-phase 

Notes

Acknowledgements

This work was funded by an Australian Research Council (ARC) Discovery Project Grant (DP160100368 to MP). MP was supported by an ARC Future Fellowship (FT120100170). LH was supported by an Australian Postgraduate Fellowship. Microscopy was performed in the Queensland Brain Institute’s Advanced Microscopy Facility.

References

  1. Arai Y, Pulvers JN, Haffner C, Schilling B, Nusslein I, Calegari F, Huttner WB (2011) Neural stem and progenitor cells shorten S-phase on commitment to neuron production. Nat Commun 2:154CrossRefPubMedPubMedCentralGoogle Scholar
  2. Calegari F, Haubensak W, Haffner C, Huttner WB (2005) Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development. J Neurosci 25:6533–6538CrossRefPubMedGoogle Scholar
  3. Harris L, Zalucki O, Gobius I, McDonald H, Osinki J, Harvey TJ, Essebier A, Vidovic D, Gladwyn-Ng I, Burne TH et al (2016) Transcriptional regulation of intermediate progenitor cell generation during hippocampal development. Development 143:4620–4630CrossRefPubMedPubMedCentralGoogle Scholar
  4. Liboska R, Ligasova A, Strunin D, Rosenberg I, Koberna K (2012) Most anti-BrdU antibodies react with 2′-deoxy-5-ethynyluridine -- the method for the effective suppression of this cross-reactivity. PLoS ONE 7:e51679CrossRefPubMedPubMedCentralGoogle Scholar
  5. Martynoga B, Morrison H, Price DJ, Mason JO (2005) Foxg1 is required for specification of ventral telencephalon and region-specific regulation of dorsal telencephalic precursor proliferation and apoptosis. Dev Biol 283:113–127CrossRefPubMedGoogle Scholar
  6. 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 18:311–318CrossRefPubMedGoogle Scholar
  7. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H et al (2008) Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132:487–498CrossRefPubMedGoogle Scholar
  8. Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci USA 105:2415–2420CrossRefPubMedPubMedCentralGoogle Scholar
  9. Schorl C, Sedivy JM (2007) Analysis of cell cycle phases and progression in cultured mammalian cells. Methods 41:143–150CrossRefPubMedPubMedCentralGoogle Scholar
  10. Takahashi T, Nowakowski RS, Caviness VS Jr (1995) The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall. J Neurosci 15:6046–6057CrossRefPubMedGoogle Scholar
  11. Tang X, Falls DL, Li X, Lane T, Luskin MB (2007) Antigen-retrieval procedure for bromodeoxyuridine immunolabeling with concurrent labeling of nuclear DNA and antigens damaged by HCl pretreatment. J Neurosci 27:5837–5844CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.The School of Biomedical Sciences, The Faculty of MedicineThe University of QueenslandBrisbaneAustralia
  2. 2.Queensland Brain Institute, The Faculty of MedicineThe University of QueenslandBrisbaneAustralia

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