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Histochemistry

, Volume 87, Issue 4, pp 331–338 | Cite as

Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration

  • T. Araki
  • A. Yamamoto
  • M. Yamada
Article

Summary

In an attempt to achieve accurate quantification of DNA levels in cell nuclie, we studied the influence of salt concentration on the fluorescence of cell nuclei complexed with Hoechst-33258 (Hoe) fluorochrome. The fluorescence of cell nuclei was compared with that of extracted DNA as well as that of nucleosome core. Conformational changes in these complexes were examined by measuring both fluorescence anisotropy and fluorescence lifetime in the nanosecond region. The results showed that the fluorescence of DNA-Hoe was quenched by the nucleosomal structure, there being an associated increase in anisotropy and a decrease in the fluorescence lifetime; however, the fluorescence was restored to the orginal level by the addition of a high concentration of NaCl, CsCl, or LiCl. The reduction in fluorescence may have been due to loss of fluorescence energy caused by collision of the fluorophore with histones in the nucleosome. The addition of 1 M NaCl to the medium used for staining with Hoe greatly stabilized the fluorescence of DNA in cell nuclei. The DNA content of individual cell nuclei was determined by comparing the fluorescence of these nuclei with that of a standard DNA solution. For lymphocytes and liver ploidy cells, reasonably accurate values were obtained by applying the present method.

Keywords

Salt Concentration Cell Nucleus LiCl CsCl Fluorescence Lifetime 
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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References

  1. Araki T, Yamada M (1985) Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ cell nuclei. Histochemistry 83:299–301Google Scholar
  2. Araki T, Tohno Y, Takakusu A, Yamada M (1985) Polarization fluorometry of nucleosome DNA structure with Hoechst 33258 fluorochrome. Cell Mol Biol 31:407–412Google Scholar
  3. Ashikawa I, Kinoshita K, Ikegami A, Nishimura Y, Tsuboi M, Watanabe K, Nakano T (1983) Internal motion of deoxyribo-nucleic acid in chromatin. Nanosecond fluorescence studies of intercalated ethidium. Biochemistry 22:6018–6026Google Scholar
  4. Brodie S, Giron J, Latt SA (1975) Estimation of accessibility of DNA in chromatin from fluorescence measurements of electron excitation energy transfer. Nature 253:470–471Google Scholar
  5. Brunk CF, Jones KC, James TW (1979) Assay for nanogram quantities of DNA in cellular homogenates. Anal Biochem 92:497–500Google Scholar
  6. Burton K (1956) A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 100:315–323Google Scholar
  7. Cesarone CF, Bolognesi C, Santi L (1979) Improved microfluorometric DNA determination in biological material using 33258 Hoechst. Anal Biochem 100:188–197Google Scholar
  8. Cowden RR, Curtis SK (1981) Microfluorometric investigations of chromatin structure I. Evaluation in nine DNA-specific fluorochromes as probes of chromatin organization. Histochemistry 72:11–23Google Scholar
  9. Downs TR, Wilfinger WW (1983) Fluorometric quantification of DNA in cells and tissue. Anal Biochem 131:538–547Google Scholar
  10. Dutt MK (1971) Intensity of Feulgen staining of rat liver nuclei fixed with two different concentrations of formalin. Histochemie 25:60–62Google Scholar
  11. Fasman GD (1976) Handbook of biochemistry and molecular biology, vol 2, 3rd edn. CRC Press, Boca Raton, FL, pp 270–272Google Scholar
  12. Ferrante A, Thong YH (1980) Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the Hypaque-Ficoll method. J Immunol Methods 36:109–117Google Scholar
  13. Higashi K, Narayanank S, Adams H, Busch H (1966) Utilization of the citric acid procedure and zonal ultracentrifugation for mass isolation of nuclear RNA from Walker 256 carcinosarcoma. Cancer Res 26:1582–1590Google Scholar
  14. Kleiman L, Huang RC (1972) Reconstitution of chromatin. The sequential binding of histones to DNA in the presence of salt and urea. J Mol Biol 64:1–8Google Scholar
  15. Labarca C, Paigen K (1980) A simple rapid and sensitive DNA assay procedure. Anal Biochem 102:334–352Google Scholar
  16. Latt SA, Wohlleb JC (1975) Optical studies of the interaction of 33258 Hoechst with DNA, chromatin, and metaphase chromosomes. Chromosoma 52:297–316Google Scholar
  17. Müller W, Gautier F (1975) Interactions of heteroaromatic compounds with nucleic acids. A.T-specific non-intercalating DNA ligands. Eur J Biochem 54:385–394Google Scholar
  18. Redi CA, Garagha S, Bottiroli G (1986) Cytochemical evaluation of sperm and lymphocyte DNA content after treatment with 5N HCl. Histochemistry 84:41–44Google Scholar
  19. Tohno Y (1983) Occurrence of intranuclear aggregations on nuclease digestion of isolated rat ascites hepatoma nuclei. Cell Mol Biol 29:539–547Google Scholar
  20. Wahl P, Paoletti J, LePecq JB (1970) Decay of fluorescence emission anisotropy of ethidium bromide-DNA complex. Evidence for an internal motion in DNA. Proc Natl Acad Sci USA 65:417–421Google Scholar
  21. Yguerabide J (1971) Nanosecond fluorescence spectroscopy of macromolecules. In: Hirs CHW, Timasheff N (eds) Methods in enzymology, vol 26. Academic Press, New York London, p 518Google Scholar
  22. Zamenhof A, Griboff G, Marullo N (1954) Studies on the resistance of deoxyribonucleic acid to physical and chemical factors. Biochim Biophys Acta 13:459–470Google Scholar

Copyright information

© Springer-Verlag 1987

Authors and Affiliations

  • T. Araki
    • 1
  • A. Yamamoto
    • 1
  • M. Yamada
    • 1
  1. 1.Laboratory for Cytochemistry, Department of Anatomy, School of MedicineTokushima UniversityTokushimaJapan

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