Cell and Tissue Biology

, Volume 6, Issue 3, pp 302–308 | Cite as

Peculiarities of cytometrical methods of DNA content determination in the nucleus

  • Yu. G. Pichugin
  • K. A. Semiyanov
  • A. V. Chernyshev
  • I. G. Palchikova
  • L. V. Omelyanchyuk
  • V. P. Maltsev
Article

Abstract

This work has proposed a new theoretical approach to analysis of histograms of DNA content, which are obtained by the method of flow cytometry, in cells of Drosophila melanogaster imaginal discs. The precision of measurements of the DNA amount in G1 and G2(M) phases has been shown to be limited by precision of instrument tuning of zero of the flow cytometer. Use of the calculative zero of the flow cytometer and of dividing cells as standards of the DNA content is able to increase severalfold the precision of the DNA measurements in nuclei of the species. Comparative analysis of errors of various methods of measurement of the DNA content in cell nuclei is also performed. For methods of flow fluorescent cytometry, confocal scanning, and cytophotometry of the Feulgen-stained nuclei, it has been shown that, at present, the mean square errors of the DNA content measurements are within the interval of values considered acceptable for biological studies (0.02 < CV < 0.06).

Keywords

fluorescent flow cytometry DNA content cell cycle 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Agroskin, L.S. and Papayan, T.V., Tsitofotometriya (Cytophotometry), Leningrad: Nauka, 1977.Google Scholar
  2. Darzynkiewicz, Z., Halicka, H.D., and Zhao, H., Analysis of Cellular DNA Content by Flow and Laser Scanning Cytometry, Adv. Exp. Med. Biol., 2010, vol. 676, pp. 137–147.PubMedCrossRefGoogle Scholar
  3. De la Cruz, A.F. and Edgar, B.A., Flow Cytometric Analysis of Drosophila Cells, Methods Mol. Biol., 2008, vol. 420, pp. 373–389.PubMedCrossRefGoogle Scholar
  4. Dean, P.N., Data Processing, in Flow Cytometry and Sorting, New York: Wiley-Liss, 1991, pp. 415–444.Google Scholar
  5. Jones, D.R., Pertunnen, C.D., and Stuckman, B.E., Lipschitzian Optimization without the Lipschitz Constant, J. Optimiz. Theor. Applic., 1993, vol. 79, pp. 157–181.CrossRefGoogle Scholar
  6. Lebedeva, L.I., Akhmametyeva, E.M., and Omelyanchuk, L.V., Dynamics of the Spatial Organization of the Chromosome Set in Cells of Drosophila melanogaste Imaginal Disks Normally and under the Action of the Tumor-Inducing Mutation Merlin, Russ. J. Genet., 2010, vol. 46, no. 2, pp. 157–163.CrossRefGoogle Scholar
  7. Lebedeva, L.I., Dubatolova, T.D., and Omelyanchuk, L.V., Fluorometric Identification of Chromatin-Packing Level beyond Resolution of Light Microscopy, Cell. Tiss. Biol., 2011, vol. 5, no. 1, pp. 98–102.CrossRefGoogle Scholar
  8. Omelyanchuk, L.V., Semeshin, V.F., Alekseeva, A.L., Pal’chikova, I.G., and Zhimulev, I.F., Integral Method for Measuring the Quantity of Cellular DNA Content by Digital Microphotography, Cell. Tiss. Biol., 2010, vol. 4, no. 3, pp. 305–308.CrossRefGoogle Scholar
  9. Puech, M. and Giroud, F., Standardisation of DNA Quantitation by Image Analysis: Quality Control of Instrumentation, Cytometry, 1999, vol. 36, pp. 11–17.PubMedCrossRefGoogle Scholar
  10. Rasch, E.M. and Wyngaard, G.A., Evidence for Endoreduplication: Germ Cell DNA Levels Prior to Chromatin Diminution in Mesocyclops edax, J. Histochem. Cytochem., 2001, vol. 49, pp. 795–796.PubMedCrossRefGoogle Scholar
  11. Rasch, E.M., Lee, C.E., and Wyngaard, G.A., DNA-Feulgen Cytophotometric Determination of Genome Size for the Freshwater-Invading Copepod Eurytemora affinis, Genome, 2004, vol. 47, pp. 559–564.PubMedCrossRefGoogle Scholar
  12. Rasch, E.M., Wyngaard, G.A., and Connelly, B.A., Heterochromatin Endoreduplication Prior to Gametogenesis and Chromatin Diminution during Early Embryogenesis in Mesocyclops edax (Copepoda: Crustacea), J. Morph., 2008, vol. 269, pp. 387–397.PubMedCrossRefGoogle Scholar
  13. Semeshin, V.F., Omelyanchuk, L.V., Alekseeva, A.L., Ivankina, E.A., Sheveleva, N.G., and Zhimulev, I.F., DNA Content in Nuclei of Cyclops kolensis and Cyclops insignis (Crustacea, Copepoda), Cell. Tiss. Biol., 2011, vol. 5, no. 3, pp. 300–304.CrossRefGoogle Scholar
  14. Stal, O. and Baldetorp, B., S-Phase Fraction Assessed by a Variant of the Rectangular Model Adapted to the Flow-Cytometric DNA Histogram Profile, Cytometry, 1998, vol. 33, pp. 487–491.PubMedCrossRefGoogle Scholar
  15. Strokotov, D.I., Yurkin, M.A., Gilev, K.V., van, Bockstaele, D.R., Hoekstra, A.G., Rubtsov, N.B., and Maltsev, V.P., Is there a Difference between T- and B-Lymphocyte Morphology?, J. Biomed. Opt., 2009, vol. 14, p. 064036.PubMedCrossRefGoogle Scholar
  16. Vindelov, L.L., Christensen, I.J., and Nissen, N.I., A Detergent-Tripsin Method for the Preparation of Nuclei for Cytometric DNA Analysis, Cytometry, 1983, vol. 3, pp. 323–327.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2012

Authors and Affiliations

  • Yu. G. Pichugin
    • 2
    • 3
  • K. A. Semiyanov
    • 4
  • A. V. Chernyshev
    • 2
    • 3
  • I. G. Palchikova
    • 3
    • 5
  • L. V. Omelyanchyuk
    • 1
    • 3
  • V. P. Maltsev
    • 2
    • 3
  1. 1.Institute of Chemical Biology and Fundamental Medicine, Siberian BranchRussian Academy of SciencesNovosibirskRussia
  2. 2.Institute of Chemical Kinetics and Combustion, Siberian BranchRussian Academy of SciencesNovosibirskRussia
  3. 3.Novosibirsk UniversityNovosibirskRussia
  4. 4.Institute of Cytology and Genetics, Siberian BranchRussian Academy of SciencesNovosibirskRussia
  5. 5.Technological Design Institute of Scientific Instrument Engineering, Siberian BranchRussian Academy of SciencesNovosibirskRussia

Personalised recommendations