Unit

UNIT 7.34 Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining

  1. Paolo Cappella1,
  2. Fabio Gasparri1,
  3. Maurizio Pulici2,
  4. Jürgen Moll1

Published Online: 1 JUL 2008

DOI: 10.1002/0471142956.cy0734s45

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Cappella, P., Gasparri, F., Pulici, M. and Moll, J. 2008. Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining. Current Protocols in Cytometry. 58:7.34:7.34.1–7.34.13.

Author Information

  1. 1

    Department of Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy

  2. 2

    Department of Chemistry, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy

Publication History

  1. Published Online: 1 JUL 2008
  2. Published Print: JUL 2008

Abstract

Determination of incorporation of the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle (see unit 7.7). However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by an alkenyl deoxyuridine (5-ethynyl-2′-deoxyuridine, EdU), which is incorporated into DNA. The nucleotide exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction (“click chemistry” coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is preferred over traditional BrdU detection since it is more sensitive and allows multicolor and multiplex analysis in FCM and imaging. Curr. Protoc. Cytom. 58:7.34.1-7.34.13. © 2011 by John Wiley & Sons, Inc.

Keywords:

  • BrdU azide;
  • BMA;
  • ethynyl deoxyuridine;
  • click chemistry;
  • cell cycle;
  • multiplex analysis;
  • chemical reporter