Click‐iT Proliferation Assay with Improved DNA Histograms

Awtar Krishan1, Ronald M. Hamelik1

1 University of Miami Miller School of Medicine, Miami, Florida
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.36
DOI:  10.1002/0471142956.cy0736s52
Online Posting Date:  April, 2010
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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

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Modified Click‐iT Proliferation Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Modified Click‐iT Proliferation Assay

  Materials
  • Cells cultured in appropriate culture medium
  • Appropriate culture medium
  • Click‐iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen, cat. no. 35002) including:
    • EdU
  • 1% (w/v) bovine serum albumin in DPBS (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS) without calcium and magnesium, pH 7.1 to 7.2 (Invitrogen)
  • 0.25% trypsin/ 0.02% EDTA solution (Invitrogen)
  • Cell lysis buffer (see recipe)
  • PI staining solution (see recipe)
  • 25‐ or 75‐cm2 flasks
  • 37°C CO 2 tissue culture incubator
  • 15‐ml (17 × 120 mm) plastic screw‐cap conical centrifuge tubes
  • 37°C water bath
  • 5‐ml (12 × 75 mm) plastic snap‐cap centrifuge tubes
  • Vortex
  • Flow cytometer equipped with 488 nm excitation source and the following filters for emission collection: 530 ± 20 nm bandpass filter for Alexa Fluor 488 and 625 ± 20 nm bandpass filter for PI
NOTE: Click‐iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit reagents are made and stored as per instructions in the kit. We prefer to make up the solutions in DMSO rather than in saline.
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Figures

  •   FigureFigure 7.36.1 Comparison of DNA histograms and EdU labeling using the standard and modified technique. A and D, cells stained by the original method using fixation with paraformaldehyde and staining of DNA with 7‐AAD. B and E, cells in suspension stained after lysis and staining with PI. C and F, adherent cells after lysis and staining with PI.
  •   FigureFigure 7.36.2 Comparison of the standard and the modified technique.

Videos

Literature Cited

Literature Cited
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   Darzynkiewicz, Z., Traganos, F., Kapuscinski, J., Staiano‐Coico, L., and Melamed, M.R. 1984. Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. Cytom. Part A 5:355‐363.
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   Krishan, A. 1990. Rapid DNA content analysis by the propidium iodide‐hypotonic citrate method. Methods Cell Biol. 33:121‐125.
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   Toba, K., Winton, E.F., and Bray, R.A. 1992. Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S‐phase fraction, and surface phenotype using single laser instrumentation. Cytom. Part A 13:60‐67.
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