Analysis of Cellular DNA Content by Flow and Laser Scanning Cytometry
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.
KeywordsMethod Cell Biol Laser Scan Cytometry Trout Erythrocyte Supravital Staining Laser Scanning Cytometer
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- 6.Bagwell BC. Theoretical aspects of flow cytometry data analysis. In: Bauer KE, Duque RE, Shankey TV, eds. Clinical Flow Cytometry: Principles and Applications. Baltimore: Williams & Wilkins, 1993:41–61.Google Scholar
- 7.Rabinovitch PS. Practical considerations for DNA content and cell cycle analysis. In: Bauer KE, Duque RE, Shankey TV, eds. Clinical Flow Cytometry: Principles and Applications. Baltimore: Williams & Wilkins, 1993:117–142.Google Scholar
- 23.Crissman HA, Steinkamp JA. Cytochemical techniques for multivariate analysis of DNA and other cell constituents. In: Melamed MR, Lindmo T, Mendelsohn ML, eds. Flow Cytometry and Sorting. New York: Wiley-Liss: 1992:227–47.Google Scholar