Critical Aspects in Analysis of Cellular DNA Content

Zbigniew Darzynkiewicz1

1 The Brander Cancer Research Institute and Department of Pathology, New York Medical Center, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.2
DOI:  10.1002/0471142956.cy0702s56
Online Posting Date:  April, 2011
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This unit covers general aspects of DNA content analysis and provides introductory or complementary information to the specific protocols of DNA content assessment in this chapter. It describes principles of DNA content analysis and outlines difficulties and pitfalls common to these methods. It also reviews methods of DNA staining in live, permeabilized, and fixed cells, and in cell nuclei isolated from paraffin‐embedded tissues, as well as the approaches to stain DNA concurrently with cell immunophenotype. This unit addresses factors affecting accuracy of DNA measurement, such as chromatin features restricting accessibility of fluorochromes to DNA, stoichiometry of interaction with DNA, and “mass action law” characterizing binding to DNA in relation to unbound fluorochrome concentration. It also describes controls to ensure accuracy and quality control of DNA content determination and principles of DNA ploidy assessment. Because many aspects of DNA content analysis are common to protocols in UNITS 7.3, 7.6, 7.16, 7.20, 7.23, & 7.25, certain parts of this unit provide information redundant with commentaries in these units. Curr. Protoc. Cytom. 56:7.2.1‐7.2.8. © 2011 by John Wiley & Sons, Inc.

Keywords: cell cycle; apoptosis; ploidy; DNA index; stoichiometry; fluorochrome; chromatin

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Table of Contents

  • Introduction
  • Supravital Cell Staining
  • Staining of DNA after Lysis of Plasma Membrane
  • DNA Staining in Fixed Cells
  • Analysis of DNA Content in Cell Nuclei Isolated from Paraffin‐Embedded Tissues
  • Concurrent Analysis of DNA Content and Cell Surface Antigen
  • Accuracy of DNA Content Measurement
  • Role of Chromatin Structure in Accessibility of DNA to Fluorochromes
  • Chemical Mass Action Law in Fluorochrome Binding to DNA
  • Assessment of DNA Ploidy
  • Literature Cited
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Literature Cited

Literature Cited
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