Unit

UNIT 7.36 Click-iT Proliferation Assay with Improved DNA Histograms

  1. Awtar Krishan,
  2. Ronald M. Hamelik

Published Online: 1 APR 2010

DOI: 10.1002/0471142956.cy0736s52

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Krishan, A. and Hamelik, R. M. 2010. Click-iT Proliferation Assay with Improved DNA Histograms. Current Protocols in Cytometry. 52:7.36:7.36.1–7.36.7.

Author Information

  1. University of Miami Miller School of Medicine, Miami, Florida

Publication History

  1. Published Online: 1 APR 2010
  2. Published Print: APR 2010

Abstract

The Click-iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated “click” reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7-aminoactinomycin D (7-AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of cells can be directly labeled using the Click-iT Alexa Fluor 488 Assay kit and stained with propidium iodide. This modified procedure using isolated nuclei and propidium iodide staining results in DNA histograms with better resolution (lower coefficient of variation of the G1 peak) and shorter processing time by eliminating the fixation and permeabilization steps. Curr. Protoc. Cytom. 52:7.36.1-7.36.7. © 2010 by John Wiley & Sons, Inc.

Keywords:

  • flow cytometry;
  • DNA content;
  • proliferation assay;
  • cell cycle;
  • Click-iT assay