Skip to main content

Cell Cycle Analysis of CML Stem Cells Using Hoechst 33342 and Propidium Iodide

  • Protocol
  • First Online:
Book cover Chronic Myeloid Leukemia

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1465))

Abstract

Chronic myeloid leukemia (CML) is a myeloproliferative disease with an expansion of white blood cells. The current treatments for CML are shown not to be long-term effective because of CML stem cells’ insensitivity to tyrosine kinase inhibitors. Therefore, studying more about CML stem cells is essential to understand the pathways of CML stem cell development and proliferation and finally lead to effective treatments to eliminate CML stem cells and eradicate CML. This chapter describes two methods to analyze cell cycle of CML stem cells. The rare population of CML stem cells can be identified by staining with cell surface markers, and then DNA-binding dyes Hoechst 33342 and propidium iodide (PI) are added to stain the DNA content which is changed when cells go through different phases of the cell cycle. Samples are run through the flow cytometer to be analyzed based on different absorbance and emission wavelengths of different florescent colors.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Spangrude GJ, Heimfeld S, Weissman IL (1988) Purification and characterization of mouse hematopoietic stem cells. Science 241:58–62

    Article  CAS  PubMed  Google Scholar 

  2. Rowley JD (1873) A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature 243:290

    Article  Google Scholar 

  3. Shtivelman E et al (1986) Alternative splicing of RNAs transcribed from the human abl gene and from the bcr-abl fused gene. Cell 47:277–284

    Article  CAS  PubMed  Google Scholar 

  4. Druker BJ, Sawyers CL, Kantarjian H et al (2001) Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 344:1038–1042

    Article  CAS  PubMed  Google Scholar 

  5. Ren R (2005) Mechanisms of BCR-ABL in the pathogenesis of chronic myelogenous leukaemia. Nat Rev Cancer 5:172–183

    Article  CAS  PubMed  Google Scholar 

  6. Graham S, Jørgensen H, Allan E et al (2002) Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI57J in vitro. Blood 99:319–325

    Article  CAS  PubMed  Google Scholar 

  7. Kantarjian H et al (2002) Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 346:645–652

    Article  CAS  PubMed  Google Scholar 

  8. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B (1992) Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A 89:2804–2808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Osawa M, Hanada K, Hamada H, Nakauchi H (1996) Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273:242–245

    Article  CAS  PubMed  Google Scholar 

  10. Okada S et al (1992) In vivo and in vitro stem cell function of c-kit- and Sca-1 positive murine hematopoietic cells. Blood 12:3044–3050

    Google Scholar 

  11. Li S et al (1999) The P190, P210 and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity. J Exp Med 189:1399–1412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Crissman HA, Hirons GT (1994) Staining of DNA in live and fixed cells. Methods Cell Biol 41:196–209

    Google Scholar 

  13. Kapinas K et al (2013) The abbreviated pluripotent cell cycle. J Cell Physiol 228:9–20

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Endl E et al (2001) Antibodies against the Ki-67 protein: assessment of the growth fraction and tools for cell cycle analysis. Methods Cell Biol 63:399–418

    Article  CAS  PubMed  Google Scholar 

  15. Lalande ME (1981) Hoechst 33342 dye uptake as a probe of membrane permeability changes in mammalian cells. Proc Natl Acad Sci U S A 78:363–367

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Pozawrowski P, Darzynkiewicz Z (2004) Analysis of cell cycle by flow cytometry. Methods Mol Biol 281:301–311

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yi Shan .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

DeSouza, N., Zhou, M., Shan, Y. (2016). Cell Cycle Analysis of CML Stem Cells Using Hoechst 33342 and Propidium Iodide. In: Li, S., Zhang, H. (eds) Chronic Myeloid Leukemia. Methods in Molecular Biology, vol 1465. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4011-0_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-4011-0_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-4009-7

  • Online ISBN: 978-1-4939-4011-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics