Russian Journal of Plant Physiology

, Volume 61, Issue 6, pp 899–909 | Cite as

Extra perspectives of 5-ethynyl-2′-deoxyuridine click reaction with fluorochrome azides to study cell cycle and deoxyribonucleoside metabolism

  • A. V. Nosov
  • A. A. Fomenkov
  • A. S. Mamaeva
  • A. E. Solovchenko
  • G. V. Novikova
Methods

Abstract

Beginning with the pioneering work of Salic and Mitchison (2008), the application of thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) for the detection of cells replicating DNA is actively expanding. Being incorporated into DNA, this nucleoside after click reaction of azide-alkyne cycloaddition with azides of fluorochromes can be easily detected by fluorescence. Recently, protocols of EdU application in combination with click reaction adapted for plant cells appeared, and they are help for a monitoring S-period of the cell cycle in the root meristems and in vitro cultured cells with the help of a microscope and flow cytometer. In this work, we focused some details of developed methods and their modifications and also recommended new protocols. In particular, we suggested combining EdU incorporation into the cells replicating DNA with subsequent isolation of protoplasts from them and their preparation for the microscopic analysis and flow cytometry. In addition, the method of determination of EdU phosphorylation dynamics in the cells in vivo is suggested.

Keywords

Arabidopsis thaliana Chlamydomonas reinhardtii Synechocystis Vigna radiata cell cycle cell culture nucleotides S-period protoplasts flow cytometry thymidine kinase fluorescence microscopy 

Abbreviations

7-AAD

7-aminoactinomycin D

BrdU

5-bromo-2′-deoxyuridine

CC

cell cycle

DAPI

4′,6-diamidino-2-phenylindol

DMSO

dimethylsulfoxide

dT

thymidine

EdU

5-ethynyl-2′-deoxyuridine

MMC

mithramycin A

PBS

phosphate-buffered saline

PI

propidium iodide

TK

thymidine kinase

TPFC

two-parameter flow cytometry

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Copyright information

© Pleiades Publishing, Ltd. 2014

Authors and Affiliations

  • A. V. Nosov
    • 1
  • A. A. Fomenkov
    • 1
  • A. S. Mamaeva
    • 1
  • A. E. Solovchenko
    • 1
    • 2
  • G. V. Novikova
    • 1
  1. 1.Timiryazev Institute of Plant PhysiologyRussian Academy of SciencesMoscowRussia
  2. 2.Faculty of BiologyMoscow State UniversityMoscowRussia

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