Abstract
Bromodeoxyuridine (BrdU) incorporation assays have long been used to detect DNA synthesis in vivo and in vitro. The key principle of this method is that BrdU incorporated as a thymidine analog into nuclear DNA represents a label that can be tracked using antibody probes. In this chapter, we describe BrdU incorporation into limbal stem cells. The colorimetric reaction produced by this assay can be detected by immunohistochemistry, and using appropriate controls, it can be used for determination of proliferating properties of restricted progenitor cells derived from the cornea.
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References
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
Cavanagh BL, Walker T, Norazit A, Meedeniya AC (2011) Thymidine analogues for tracking DNA synthesis. Molecules 16:7980–7993
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
Hoshino T, Nagashima T, Murovic J, Levin EM, Levin VA, Rupp SM (1985) Cell kinetic studies of in situ human brain tumors with bromodeoxyuridine. Cytometry 6:627–632
Rakic P (2002) Neurogenesis in adult primate neocortex: an evaluation of the evidence. Nat Rev Neurosci 3:65–71
Nowakowski RS, Hayes NL (2000) New neurons: extraordinary evidence or extraordinary conclusion? Science 288:771
Kadar T, Horwitz V, Sahar R, Cohen M, Cohen L, Gez R et al (2011) Delayed loss of corneal epithelial stem cells in a chemical injury model associated with limbal stem cell deficiency in rabbits. Curr Eye Res 36:1098–1107
Chen W, Hara K, Tian Q, Zhao K, Yoshitomi T (2007) Existence of small slow-cycling Langerhans cells in the limbal basal epithelium that express ABCG2. Exp Eye Res 84:626–634
Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105:2415–2420
Buck SB, Bradford J, Gee KR, Agnew BJ, Clarke ST, Salic A (2008) Detection of S-phase cell cycle progression using 5-ethynyl-2¢-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2¢-deoxyuridine antibodies. Biotechniques 44:927–929
Diermeier-Daucher S, Clarke ST, Hill D, Vollmann-Zwerenz A, Bradford JA, Brockhoff G (2009) Cell type specific applicability of 5-ethynyl-2¢-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry A 75:535–546
Qu D, Wang G, Wang Z, Zhou L, Chi W, Cong S et al (2011) 5-Ethynyl-2¢-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells. Anal Biochem 417:112–121
Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H (1984) Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:1710–1715
Acknowledgments
This work was supported by DOD grant W81XWH-09-1-0674 (Project 2.2), a career award and unrestricted funds from Research to Prevent Blindness and NIH core grant P30-EY14801.
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Crane, A.M., Bhattacharya, S.K. (2013). The Use of Bromodeoxyuridine Incorporation Assays to Assess Corneal Stem Cell Proliferation. In: Wright, B., Connon, C. (eds) Corneal Regenerative Medicine. Methods in Molecular Biology, vol 1014. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-432-6_4
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DOI: https://doi.org/10.1007/978-1-62703-432-6_4
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Publisher Name: Humana Press, Totowa, NJ
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