Click-iT proliferation assay with improved DNA histograms

Awtar Krishan, Ronald M. Hamelik

Research output: Contribution to journalReview articlepeer-review

5 Scopus citations

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 G 1 peak) and shorter processing time by eliminating the fixation and permeabilization steps. Curr. Protoc. Cytom. 52:7.36.1-7.36.7.

Original languageEnglish (US)
Pages (from-to)7.36.1-7.36.7
JournalCurrent Protocols in Cytometry
Issue numberSUPPL. 52
DOIs
StatePublished - Apr 1 2010

Keywords

  • Cell cycle
  • Click-iT assay
  • DNA content
  • Flow cytometry
  • Proliferation assay

ASJC Scopus subject areas

  • Medical Laboratory Technology
  • Biochemistry
  • Histology

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