Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data

Nature Protocols volume 2, pages 2057–2067 (2007)Cite this article

Abstract

Cellular proliferation is an essential feature of the adaptive immune response. The introduction of the division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has made it possible to monitor the number of cell divisions during proliferation and to examine the relationship between proliferation and differentiation. Although qualitative examination of CFSE data may be useful, substantially more information about division and death rates can be extracted from quantitative CFSE time-series experiments. Quantitative methods can reveal in detail how lymphocyte proliferation and survival are regulated and altered by signals such as those received from co-stimulatory molecules, drugs and genetic polymorphisms. In this protocol, we present a detailed method for examining time-series data using graphical and computer-based procedures available to all experimenters.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Analysis of FACS data.
Figure 2: Quantitative analysis of proliferation data.
Figure 3: Experimental situations.
Figure 4: Caveats to quantitative analysis.

Similar content being viewed by others

References

  1. Denizot, F. & Lang, R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 89, 271–277 (1986).

    Article  CAS  PubMed  Google Scholar 

  2. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63 (1983).

    Article  CAS  PubMed  Google Scholar 

  3. Gett, A.V. & Hodgkin, P.D. A cellular calculus for signal integration by T cells. Nat. Immunol. 1, 239–244 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Good, K.L., Bryant, V.L. & Tangye, S.G. Kinetics of human B cell behavior and amplification of proliferative responses following stimulation with IL-21. J. Immunol. 177, 5236–5247 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Deenick, E.K., Gett, A.V. & Hodgkin, P.D. Stochastic model of T cell proliferation: a calculus revealing IL-2 regulation of precursor frequencies, cell cycle time, and survival. J. Immunol. 170, 4963–4972 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Hommel, M. & Hodgkin, P.D. TCR affinity promotes CD8+ T cell expansion by regulating survival. J. Immunol. 179, 2250–2260 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. De Boer, R.J. & Perelson, A.S. Estimating division and death rates from CFSE data. J. Comp. Appl. Math. 184, 104–164 (2005).

    Article  Google Scholar 

  8. De Boer, R.J., Ganusov, V.V., Milutinovic, D., Hodgkin, P.D. & Perelson, A.S. Estimating lymphocyte division and death rates from CFSE data. Bull. Math. Biol. 68, 1011–1031 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Pilyugin, S.S., Ganusov, V.V., Murali-Krishna, K., Ahmed, R. & Antia, R. The rescaling method for quantifying the turnover of cell populations. J. Theor. Biol. 225, 275–283 (2003).

    Article  PubMed  Google Scholar 

  10. Hawkins, E.D., Turner, M.L., Dowling, M.R., van Gend, C. & Hodgkin, P.D. A model of immune regulation as a consequence of randomized lymphocyte division and death times. Proc. Natl. Acad. Sci. USA 104, 5032–5037 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Cory, S., Huang, D.C. & Adams, J.M. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 22, 8590–8607 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Hodgkin, P.D., Lee, J.H. & Lyons, A.B. B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med. 184, 277–281 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Gett, A.V. & Hodgkin, P.D. Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation. Proc. Natl. Acad. Sci. USA 95, 9488–9493 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Quah, B.J.C., Warren, H.S. & Parish, C.R. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). Nat. Protoc. 2, 2049–2056 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Ganusov, V.V. et al. Quantifying cell turnover using CFSE data. J. Immunol. Methods 298, 183–200 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Lyons, A.B. & Parish, C.R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137 (1994).

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Author notes

  1. Edwin D Hawkins and Mirja Hommel: These authors contributed equally to this work.

Authors and Affiliations

  1. Immunology Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3050, Victoria, Australia

    Edwin D Hawkins, Mirja Hommel, Marian L Turner, Francis L Battye, John F Markham & Philip D Hodgkin

  2. Department of Medical Biology, University of Melbourne, Melbourne, 3010, Victoria, Australia

    Edwin D Hawkins & Marian L Turner

Authors
  1. Edwin D Hawkins
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mirja Hommel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marian L Turner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Francis L Battye
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John F Markham
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Philip D Hodgkin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Philip D Hodgkin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hawkins, E., Hommel, M., Turner, M. et al. Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat Protoc 2, 2057–2067 (2007). https://doi.org/10.1038/nprot.2007.297

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.297

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing