Advertisement

Synchronization of Mammalian Cells and Nuclei by Centrifugal Elutriation

  • Gaspar BanfalviEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 761)

Abstract

Synchronized populations of large numbers of cells can be obtained by centrifugal elutriation on the basis of sedimentation properties of small round particles, with minimal perturbation of cellular functions. The physical characteristics of cell size and sedimentation velocity are operative in the technique of centrifugal elutriation also known as counterstreaming centrifugation. The elutriator is an advanced device for increasing the sedimentation rate to yield enhanced resolution of cell separation. A random population of cells is introduced into the elutriation chamber of an elutriator rotor running in a specially designed centrifuge. By increasing step by step the flow rate of the elutriation fluid, successive populations of relatively homogeneous cell size can be removed from the elutriation chamber and used as synchronized subpopulations. For cell synchronization by centrifugal elutriation early log S phase cell populations are most suitable where most of the cells are in G1 and S phase (>80%). Protocols for the synchronization of nuclei of murine pre-B cells and high-resolution centrifugal elutriation of CHO cells are given. The verification of purity and cell cycle positions of cells in elutriated fractions includes the measurement of DNA synthesis by [3H]-thymidine incorporation and DNA content by propidium iodide flow cytometry.

Key words

Counterstreaming centrifugation cell separation velocity sedimentation elutriator resolution power 

Notes

Acknowledgment

This work was supported by the OTKA grant TO42762 (G.B.).

References

  1. 1.
    Lindahl, P. E. (1948) Principle of a counter-streaming centrifuge for the separation of particles of different sizes. Nature 161, 648–649.PubMedCrossRefGoogle Scholar
  2. 2.
    McEwen, C. R., Stallard, R. W., and Juhos, E. T. (1968) Separation of biological particles by centrifugal elutriation. Anal. Biochem. 23, 369–377.PubMedCrossRefGoogle Scholar
  3. 3.
    Sanderson, R. J., Bird, K. E., Palmer, N. F., and Brenman, J. (1976) Design principles for a counterflow centrifugation cell separation chamber. Appendix: a derivation of the equation of motion of a particle under combined centrifugal and hydrodynamic fields. Anal. Biochem. 71, 615–622.PubMedCrossRefGoogle Scholar
  4. 4.
    Pretlow, T. G. II, and Pretlow, T. P. (1979) Centrifugal elutriation (counterstreaming centrifugation) of cells. Cell Biophys. 1, 195–210.PubMedCrossRefGoogle Scholar
  5. 5.
    Meistrich, M. L. (1983) Experimental factors involved in separation by centrifugal elutriation. In: Pretlow, T. G. II, Pretlow, T. P. (eds.) Cell separation, Vol. 2, pp. 33–61. Academic, New York, NY.Google Scholar
  6. 6.
    Childs, G. V., Lloyd, J. M., Unabia, G., and Rougeau, D. (1988) Enrichment of corticotropes by counterflow centrifugation. Endocrinology 123, 2885–2895.PubMedCrossRefGoogle Scholar
  7. 7.
    Kauffman, M. G., Noga, S. J., Kelly, T. J., and Donnenberg, A. D. (1990) Isolation of cell cycle fractions by counterflow centrifugal elutriation. Anal. Biochem. 191, 41–46.PubMedCrossRefGoogle Scholar
  8. 8.
    Bauer, J. (1999) Advances in cell separation: recent developments in counterflow centrifugal elutriation and continuous flow cell separation. J. Chromatogr. B Biomed. Sci. Appl. 722, 55–69.PubMedCrossRefGoogle Scholar
  9. 9.
    Chianea, T., Assidjo, N. E., and Cardot, P. J. P. (2000) Sedimentation field-flow-fractionation: emergence of a new cell separation methodology. Talanta 51, 835–847.PubMedCrossRefGoogle Scholar
  10. 10.
    Uzbekov, R. E. (2004) Analysis of the cell cycle and a method employing synchronized cells for study of protein expression at various stages of the cell cycle. Biochemistry 69, 485–496.PubMedGoogle Scholar
  11. 11.
    Banfalvi, G. (2008) Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation. Nat. Protoc. 3, 663–673.PubMedCrossRefGoogle Scholar
  12. 12.
    Keng, P. C., Li, C. K., and Wheeler, K. T. (1980) Synchronization of 9L rat brain tumor cells by centrifugal elutriation. Cell Biophys. 2, 191–206.PubMedGoogle Scholar
  13. 13.
    Riccardi, C., and Nicoletti, I. (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protoc. 1, 1458–1461.PubMedCrossRefGoogle Scholar
  14. 14.
    Doleel, J., Greilhuber, J., and Suda, J. (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat. Protoc. 2, 2233–2244.CrossRefGoogle Scholar
  15. 15.
    Terry, N. H. A., and White, R. A. (2006) Flow cytometry after bromodeoxyuridine labeling to measure S and G2.M phase durations plus doubling times in vitro and in vivo. Nat. Protoc. 1, 859–869.PubMedCrossRefGoogle Scholar
  16. 16.
    Schmid, I., Uittenbogaart, C., and Jamieson, B. D. (2006) Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-aminoactinomycin D. Nat. Protoc. 1, 187–190.CrossRefGoogle Scholar
  17. 17.
    Mukhopadhyay, P., Rajesh, M., Haskó, G., Hawkins, B. J., Madesh, M., and Pacher, P. (2007) Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nat. Protoc. 2, 2295–2301.PubMedCrossRefGoogle Scholar
  18. 18.
    Ferlini, C., and Scambia, G. (2007) Assay for apoptosis using the mitochondrial probes, Rhodamine123 and 10-N-nonyl acridine orange. Nat. Protoc. 2, 3111–3114.PubMedCrossRefGoogle Scholar
  19. 19.
    van Genderen, H., Kenis, H., Lux, P., Ungeth, L., Maassen, C., Deckers, N., Narula, J., Hofstra, L., and Reutelingsperger, C. (2006) In vitro measurement of cell death with the annexin A5 affinity assay. Nat. Protoc. 1, 363–367.PubMedCrossRefGoogle Scholar
  20. 20.
    Quah, B. J. C., Warren, H. S., and Parish, C. R. (2007) Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat. Protoc. 2, 2049–2056.PubMedCrossRefGoogle Scholar
  21. 21.
    Chattopadhyay, P. K., Yu, J., and Roederer, M. (2006) Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression Nat. Protoc. 1, 1–6.PubMedCrossRefGoogle Scholar
  22. 22.
    Pittet, M. J., Swirski, F. K., Reynolds, F., Josephson, L., and Weissleder, R. (2006) Labeling of immune cells for in vivo imaging using magnetofluorescent nanoparticles. Nat. Protoc. 1, 73–79.PubMedCrossRefGoogle Scholar
  23. 23.
    Offer, H., Zurer, I., Banfalvi, G., Rehak, M., Falcovitz, A., Milyavsky, M., Goldfinger, N., and Rotter, V. (2001) p53 modulates base excision repair activity in a cell cycle specific manner following genotoxic stress. Cancer Res. 61, 88–96.PubMedGoogle Scholar
  24. 24.
    Basnakian, A., Banfalvi, G., and Sarkar, N. (1989) Contribution of DNA polymerase delta to DNA replication in permeable CHO cells synchronized in S phase. Nucleic Acids Res. 17, 4757–4767.PubMedCrossRefGoogle Scholar
  25. 25.
    Banfalvi, G., Nagy, G., Gacsi, M., Roszer, T., and Basnakian, A. (2006) Common pathway of chromatin condensation in mammalian cells. DNA Cell Biol. 25, 295–301.PubMedCrossRefGoogle Scholar
  26. 26.
    Rehak, M., Csuka, I., Szepessy, E., and Banfalvi, G. (2000) Subphases of DNA replication in Drosophila cells. DNA Cell Biol. 19, 607–612.PubMedCrossRefGoogle Scholar
  27. 27.
    Banfalvi, G. (2006) Structure of interphase chromosomes in the nuclei of Drosophila cells. DNA Cell Biol. 25, 547–553.PubMedCrossRefGoogle Scholar
  28. 28.
    Banfalvi, G., Littlefield, N., Hass, B., Mikhailova, M., Csuka, I., Szepessy, E., and Chou, W. M. (2000) Effect of cadmium on the relationship between replicative and repair DNA synthesis in synchronized CHO cells. Eur. J. Biochem. 267, 6580–6585.PubMedCrossRefGoogle Scholar
  29. 29.
    Coulter, W. H. (1957) High speed automatic blood cell counter and cell size analyzer. Proc. Natl. Electron. Conf. 12, 1034–1040.Google Scholar
  30. 30.
    Banfalvi, G., Mikhailova, M., Poirier, L. A., and Chou, M. W. (1997) Multiple subphases of DNA replication in CHO cells. DNA Cell Biol. 16, 1493–1498.PubMedCrossRefGoogle Scholar
  31. 31.
    Banfalvi, G., Poirier, A. L., Mikhailova, M., and Chou, W. M. (1997) Relationship of repair and replicative DNA synthesis to cell cycle in Chinese hamster Ovary (CHO-K1) cells. DNA Cell Biol. 16, 1155–1160.PubMedCrossRefGoogle Scholar
  32. 32.
    Szepessy, E., Nagy, G., Jenei, Z., Serfozo, Z., Csuka, I., James, J., and Banfalvi, G. (2003) Multiple subphases of DNA repair and poly(ADP-ribose) synthesis in Chinese hamster ovary (CHO-K1) cells. Eur. J. Cell Biol. 82, 201–207.PubMedCrossRefGoogle Scholar
  33. 33.
    Banfalvi, G., Ujvarosi, K., Trencsenyi, G., Somogyi, C., Nagy, G., and Basnakian, A. G. (2007) Cell culture dependent toxicity and chromatin changes upon cadmium treatment in murine pre-B cells. Apoptosis 12, 1219–1228.PubMedCrossRefGoogle Scholar
  34. 34.
    Doleel, J., Greilhuber, J., and Suda, J. (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat. Protoc. 2, 2233–2244.CrossRefGoogle Scholar
  35. 35.
    Guidozzi, F. (1997) Enrichment of ovarian cancer cell suspensions by centrifugal elutriation after density gradient purification. Int. J. Gynecol. Cancer 7, 100–105.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Microbial Biotechnology and Cell BiologyUniversity of DebrecenDebrecenHungary

Personalised recommendations