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Bromodeoxyuridine (BrdU) in adult neurogenesis research - Part 2

Apr 14, 2017

Prefer to listen to the BrdU in adult neurogenesis research story?

Check out the podcast version of our two blog articles. You will learn how BrdU was established as a popular tool for neurogenesis research as well as gain insight into the latest BrdU related findings. Finally, you will go away with 5 practical tips for controlling your BrdU labeling experiments. Spend your experimental break wisely and get some science on the go in less than 15 minutes.

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Neurons In the first part of our series on BrdU in adult neurogenesis research, we described the indelible role of BrdU as a lineage tracer in neurogenesis studies. Despite its benefits, as with every treatment, there are limitations and unwanted side effects. In this post, we describe “the dark side of BrdU” and how you can carefully control your BrdU labeling experiments (Lehner et al. 2011).

It has been known since the late 1970s that incorporation of BrdU into DNA induces conformational changes in its structure, which could potentially induce harmful effects such as altering cell cycle progression (Goz 1977). Despite these findings, early studies on adult neurogenesis did not consider these possible side effects of BrdU. However, more recent studies have indicated that BrdU may have other detrimental effects than those previously described on neuronal precursors and adult brain cells.

One such study by Lehner et al. (2011) systematically demonstrates the effects of BrdU on the proliferation, cell cycle, cell death and differentiation of adult neuronal progenitor cells (NPCs). Using NPCs isolated from adult rats, the study demonstrated that BrdU inhibited NPC expansion and induced cell cycle arrest at the G0/G1 phase (Lehner et al. 2011). BrdU also increased cell death and suppressed neuronal and oligodendroglial differentiation, although astroglial fate was not affected. However, when rats were injected with BrdU, there was no effect on the proliferation of NPCs or neurogenesis at commonly used concentrations.

Other studies on embryonic stem cell derived neural stem cells (NSCs) showed that exposure to BrdU resulted in loss of global DNA methylation (Schneider and d’Adda di Fagagna 2012). Demethylation can lead to activation of affected genes, which could initiate unwanted systemic outcomes. BrdU also induced differentiation of NSCs into astrocytes in the absence of cytokines or factors known to promote such lineage differentiation (Schneider and d’Adda di Fagagna 2012). In these NSCs however, BrdU did not induce toxicity. BrdU treatment of murine NSCs isolated from the mouse forebrain also demonstrated similar results in terms of global demethylation and loss of stemness (Schneider and d’Adda di Fagagna 2012).

Duque and Rakic (2011) also expanded these findings to demonstrate the side effects of BrdU use in neurogenesis studies in primates. BrdU incorporation into the genes of adult macaque monkey brain cells resulted in significantly reduced labeled cells and cell survival compared to treatment with [3H] thymidine (Duque and Rakic 2011). These differences could be attributed to differences in the structures of BrdU and thymidine. Because BrdU contains the bromine atom, its incorporation into DNA could result in mutations that ultimately result in phenotypical changes of the BrdU treated cells.

In summary, the presented studies tell a cautionary tale that relates to interpreting results derived from the use of BrdU in neurogenesis studies, and emphasize the importance of including controls in your experiments to account for off-target effects. It should be noted that like BrdU, the performance of most cellular treatments may induce side effects, particularly at high concentrations. However, when controlled properly, BrdU is an effective tool for measuring neurogenesis and is extensively used in this area of neuroscience research (Taupin 2007). BrdU labeling is also still a popular method in cancer research (see previous blog post). Below are our top 5 tips for controlling your BrdU labeling experiments.


5 Top Tips for BrdU Labeling Experiments

  1. Include samples treated with the solvent you reconstituted the BrdU in as a negative control. For example, experiments using BrdU dissolved in ethanol or dimethyl sulfoxide (DMSO) should include samples treated with ethanol or DMSO only. Please also ensure that you add the same amounts of solvent and BrdU to your samples
  2. Titrate the BrdU reagent and antibody to identify an optimal concentration for your specific sample type and to reduce the likelihood of unwanted side effects
  3. Optimize BrdU incubation time for your sample type
  4. For assessing the impact of BrdU on loss of DNA methylation in your samples, the DNA methylation inhibitor drug 5-azacytidine (azacitidine) can be used as a positive control (Schneider and d’Adda di Fagagna 2012)
  5. To assess the impact of BrdU on cell differentiation, cells treated with fetal calf serum (FCS) can be used as a positive control for neural stem cell studies (Lehner et al. 2011)

For more experimental design and control tips, explore our wide range of control resources for applications such as flow cytometry, immunoassays, immunohistochemistry and western blotting.

Also, check out our mouse anti-BrdU antibody (clone Bu20a) that has been used in several neuroscience studies and is suitable for flow cytometric analysis of BrdU incorporation.


  • Duque A and Rakic P (2011). Different effects of bromodeoxyuridine and [3H] thymidine incorporation into DNA on cell proliferation, position, and fate. J Neurosci 31, 15205-15217.
  • Goz B (1977). The effects of incorporation of 5-halogenated deoxyuridines into the DNA of eukaryotic cells. Pharmacol Rev 29, 249-272.
  • Lehner B et al. (2011). The dark side of BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation and survival. Cell Tissue Res 345, 313-328.
  • Schneider L and d’Adda di Fagagna F (2012). Neural stem cells exposed to BrdU lose their global DNA methylation and undergo astrocytic differentiation. Nucleic Acids Res 40, 5332-5342.
  • Taupin P (2007). BrdU immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validation. Brain Res Rev 53, 198-214.

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