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:

Flow cytometry and FISH to measure the average length of telomeres (flow FISH)

Abstract

Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in combination with fluorescence measurements by flow cytometry (flow FISH). Flow FISH analysis can be performed using commercially available flow cytometers, and has the unique advantage over other methods for measuring telomere length of providing multi-parameter information on the length of telomere repeats in thousands of individual cells. The accuracy and reproducibility of the measurements is augmented by the automation of most pipetting (aspiration and dispensing) steps, and by including an internal standard (control cells) with a known telomere length in every tube. The basic protocol for the analysis of nucleated blood cells from 22 different individuals takes about 12 h spread over 2–3 days.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Example of flow FISH data analysis of nucleated blood cells from a normal human donor (83 years old).
Figure 2: Calculation of telomere length from flow FISH data.

Similar content being viewed by others

References

  1. Moyzis, R.K. et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc. Natl. Acad. Sci. USA 85, 6622–6626 (1988).

    Article  CAS  Google Scholar 

  2. Smogorzewska, A. & de Lange, T. Regulation of telomerase by telomeric proteins. Annu. Rev. Biochem. 73, 177–208 (2004).

    Article  CAS  Google Scholar 

  3. Blackburn, E.H. Switching and signaling at the telomere. Cell 106, 661–673 (2001).

    Article  CAS  Google Scholar 

  4. d'Adda di Fagagna, F., Teo, S.H. & Jackson, S.P. Functional links between telomeres and proteins of the DNA-damage response. Genes Dev. 18, 1781–1799 (2004).

    Article  Google Scholar 

  5. Harley, C.B., Futcher, A.B. & Greider, C.W. Telomeres shorten during ageing of human fibroblasts. Nature 345, 458–460 (1990).

    Article  CAS  Google Scholar 

  6. Chang, S., Khoo, C.M., Naylor, M.L., Maser, R.S. & DePinho, R.A. Telomere-based crisis: functional differences between telomerase activation and ALT in tumor progression. Genes Dev. 17, 88–100 (2003).

    Article  CAS  Google Scholar 

  7. Valdes, A.M. et al. Obesity, cigarette smoking, and telomere length in women. Lancet 366, 662–664 (2005).

    Article  CAS  Google Scholar 

  8. Collins, K. & Mitchell, J.R. Telomerase in the human organism. Oncogene 21, 564–579 (2002).

    Article  CAS  Google Scholar 

  9. Fogarty, P.F. et al. Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA. Lancet 362, 1628–1630 (2003).

    Article  CAS  Google Scholar 

  10. Vulliamy, T. et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 413, 432–435 (2001).

    Article  CAS  Google Scholar 

  11. Yamaguchi, H. et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N. Engl. J. Med. 352, 1413–1424 (2005).

    Article  CAS  Google Scholar 

  12. Harley, C.B. & Kim, N.W. Telomerase and cancer. Important Adv. Oncol. 57–67 (1996).

  13. Shay, J.W. & Roninson, I.B. Hallmarks of senescence in carcinogenesis and cancer therapy. Oncogene 23, 2919–2933 (2004).

    Article  CAS  Google Scholar 

  14. Stewart, S.A. & Weinberg, R.A. Telomeres: cancer to human aging. Annu. Rev. Cell Dev. Biol. (2006).

  15. de Lange, T. et al. Structure and variability of human chromosome ends. Mol. Cell Biol. 10, 518–527 (1990).

    Article  CAS  Google Scholar 

  16. Cawthon, R.M., Smith, K.R., O'Brien, E., Sivatchenko, A. & Kerber, R.A. Association between telomere length in blood and mortality in people aged 60 years or older. Lancet 361, 393–395 (2003).

    Article  CAS  Google Scholar 

  17. Baird, D.M., Rowson, J., Wynford-Thomas, D. & Kipling, D. Extensive allelic variation and ultrashort telomeres in senescent human cells. Nature Genet. 33, 203–207 (2003).

    Article  CAS  Google Scholar 

  18. Lansdorp, P.M. et al. Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet. 5, 685–691 (1996).

    Article  CAS  Google Scholar 

  19. Egholm, M. et al. PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566–568 (1993).

    Article  CAS  Google Scholar 

  20. Blasco, M.A. et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34 (1997).

    Article  CAS  Google Scholar 

  21. Martens, U.M. et al. Short telomeres on human chromosome 17p. Nature Genet. 18, 76–80 (1998).

    Article  CAS  Google Scholar 

  22. Zijlmans, J.M. et al. Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats. Proc. Natl. Acad. Sci. USA 94, 7423–7428 (1997).

    Article  CAS  Google Scholar 

  23. Rufer, N., Dragowska, W., Thornbury, G., Roosnek, E. & Lansdorp, P.M. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nature Biotechnol. 16, 743–747 (1998).

    Article  CAS  Google Scholar 

  24. Rufer, N. et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. J. Exp. Med. 190, 157–167 (1999).

    Article  CAS  Google Scholar 

  25. Baerlocher, G.M., Mak, J., Roth, A., Rice, K.S. & Lansdorp, P.M. Telomere shortening in leukocyte subpopulations from baboons. J. Leukoc. Biol. 73, 289–296 (2003).

    Article  CAS  Google Scholar 

  26. Ding, H. et al. Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein. Cell 117, 873–886 (2004).

    Article  CAS  Google Scholar 

  27. Brummendorf, T.H., Maciejewski, J.P., Mak, J., Young, N.S. & Lansdorp, P.M. Telomere length in leukocyte subpopulations of patients with aplastic anemia. Blood 97, 895–900 (2001).

    Article  CAS  Google Scholar 

  28. Brummendorf, T.H. et al. Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry. Blood 95, 1883–1890 (2000).

    CAS  PubMed  Google Scholar 

  29. Rufer, N. et al. Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation. Blood 97, 575–577 (2001).

    Article  CAS  Google Scholar 

  30. Awaya, N. et al. Telomere shortening in hematopoietic stem cell transplantation: a potential mechanism for late graft failure? Biol. Blood Marrow Transplant 8, 597–600 (2002).

    Article  Google Scholar 

  31. Baerlocher, G.M. & Lansdorp, P.M. Telomere length measurements in leukocyte subsets by automated multicolor flow FISH. Cytometry A 55, 1–6 (2003).

    Article  Google Scholar 

  32. Baerlocher, G.M. & Lansdorp, P.M. Telomere length measurements using fluorescence in situ hybridization and flow cytometry. Methods Cell Biol. 75, 719–750 (2004).

    Article  CAS  Google Scholar 

  33. Ly, H. et al. Functional characterization of telomerase RNA variants found in patients with hematologic disorders. Blood 105, 2332–2339 (2005).

    Article  CAS  Google Scholar 

  34. Ly, H. et al. Identification and functional characterization of two variant alleles of the telomerase RNA template gene (TERC) in a patient with Dyskeratosis Congenita. Blood 105 (6): 2332–9 (2005).

    Article  CAS  Google Scholar 

  35. Baerlocher, G.M., Mak, J., Tien, T. & Lansdorp, P.M. Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls. Cytometry 47, 89–99 (2002).

    Article  CAS  Google Scholar 

  36. Wieser, M. et al. Nuclear flow FISH: isolation of cell nuclei improves the determination of telomere lengths. Exp. Gerontol. 41, 230–235 (2006).

    Article  CAS  Google Scholar 

  37. Rufer, N., Reichenbach, P. & Romero, P. Methods for the ex vivo characterization of human CD8+ T subsets based on gene expression and replicative history analysis. Methods Mol. Med. 109, 265–284 (2005).

    CAS  PubMed  Google Scholar 

  38. Potter, A.J. & Wener, M.H. Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow FISH-DDD) to determine telomere length dynamics in proliferating cells. Cytometry A 68, 53–58 (2005).

    Article  Google Scholar 

  39. Norrback, K.F. et al. Telomerase regulation and telomere dynamics in germinal centers. Eur. J. Haematol. 67, 309–317 (2001).

    Article  CAS  Google Scholar 

  40. Martens, U.M. et al. Telomere maintenance in human B lymphocytes. Br. J. Haematol. 119, 810–818 (2002).

    Article  CAS  Google Scholar 

  41. Law, H. & Lau, Y. Validation and development of quantitative flow cytometry-based fluorescence in situ hybridization for intercenter comparison of telomere length measurement. Cytometry 43, 150–153 (2001).

    Article  CAS  Google Scholar 

  42. Hultdin, M. et al. Telomere analysis by fluorescence in situ hybridization and flow cytometry. Nucleic Acids Res. 26, 3651–3656 (1998).

    Article  CAS  Google Scholar 

  43. Derradji, H., Bekaert, S., Van Oostveldt, P. & Baatout, S. Comparison of different protocols for telomere length estimation by combination of quantitative fluorescence in situ hybridization (Q-FISH) and flow cytometry in human cancer cell lines. Anticancer Res. 25, 1039–1050 (2005).

    CAS  PubMed  Google Scholar 

  44. Brando, B. et al. Determination of telomere length by flow-fluorescence in situ hybridization in Down's syndrome patients. Int. J. Tissue React. 26, 61–64 (2004).

    CAS  PubMed  Google Scholar 

  45. Batliwalla, F.M., Damle, R.N., Metz, C., Chiorazzi, N. & Gregersen, P.K. Simultaneous flow cytometric analysis of cell surface markers and telomere length: analysis of human tonsilar B cells. J. Immunol. Methods 247, 103–109 (2001).

    Article  CAS  Google Scholar 

  46. Bartolovic, K. et al. Clonal heterogeneity in growth kinetics of CD34+CD38- human cord blood cells in vitro is correlated with gene expression pattern and telomere length. Stem Cells 23, 946–957 (2005).

    Article  CAS  Google Scholar 

  47. Roos, D. & Loos, J.A. Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes. I. Stimulation by phytohaemagglutinin. Biochim. Biophys. Acta 222, 565–582 (1970).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a US National Institutes of Health grant, the Swiss National Science Foundation, the Bernese Cancer League, Switzerland, and the National Cancer Institute of Canada with funds from the Terry Fox Run. We thank Dirk Roos (Sanquin Research at CLB, Amsterdam, The Netherlands) for advice on the ammonium chloride lysis of red blood cells, and all current and past members of the Lansdorp laboratory for their various contributions to the development of the flow FISH protocol.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Gabriela M Baerlocher or Peter M Lansdorp.

Ethics declarations

Competing interests

G.J. and P.L. are founding shareholders in Repeat Diagnostic Inc., a company specialized in leukocyte telomere length measurements using flow FISH.

Supplementary information

Supplementary Figure 1

Calibration for linearity of the flow cytometer in the fluorescence 1 channel (Fl1) using premixed MESF fluorescence beads. (PDF 4349 kb)

Supplementary Figure 2

Example of a flow FISH data acquisition template showing human nucleated blood cells hybridized in the presence of fluorescein-labelled PNA that were stained with antibodies CD45RA-Cy5 and CD57-PE and counterstained with LDS751. (PDF 7778 kb)

Supplementary Video 1

Example of Flow Cytometric Analysis (WMV 7276 kb)

Supplementary Video 2

Flow FISH in Fume Hood (WMV 3089 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Baerlocher, G., Vulto, I., de Jong, G. et al. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc 1, 2365–2376 (2006). https://doi.org/10.1038/nprot.2006.263

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.263

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

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