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Analysis of Cellular DNA Content by Flow and Laser Scanning Cytometry

  • Zbigniew Darzynkiewicz
  • H. Dorota Halicka
  • Hong Zhao
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 676)

Abstract

This chapter covers several aspects of methodology of DNA content analysis in individual cells that is most commonly used for assessment of DNA ploidy and for enumeration of cells in particular phases of the cell cycle. Briefly presented are general principles of instrumentation and cell analysis by flow- and laser scanning- cytometry. Described are major methods designed to stain DNA with fluorochromes in live cells, in detergent-permeabilized cells, in cells fixed prior to DNA staining as well as in nuclei of cells isolated from paraffin-embedded tissues. Briefly addressed are approaches to estimate cellular DNA content in conjunction with cellular immunophenotype. Discussed are factors that affect accuracy of DNA content measurement such as: (i) differences in chromatin structure of the analyzed cells that restrict DNA accessibility to fluorochromes, (ii) stoichiometry of interaction between fluorochromes and DNA in chromatin and (iii) chemical mass action law defining dependency of fluorochrome binding to DNA in relation to fluorochrome concentration and number of potential binding sites in a sample. Described also are controls used to ensure accuracy of DNA ploidy determination, the principles in ploidy assessment and possible pitfalls in analysis.

Keywords

Method Cell Biol Laser Scan Cytometry Trout Erythrocyte Supravital Staining Laser Scanning Cytometer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Landes Bioscience and Springer Science+Business Media 2010

Authors and Affiliations

  • Zbigniew Darzynkiewicz
    • 1
  • H. Dorota Halicka
    • 1
  • Hong Zhao
  1. 1.Brander Cancer Research Institute at NYMCDepartment of PathologyValhallaUSA

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