Skip to main content
SpringerLink
Log in

Stem Cell Identification by DyeCycle Violet Side Population Analysis

  • Protocol
  • First Online:
Basic Cell Culture Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 946))

Abstract

Hoechst side population (SP) analysis remains a critical technique for identifying stem cell and progenitor populations in hematopoietic and non-hematopoietic tissues, as well as potential cancer stem cells. More recently, DyeCycle Violet (DCV), a DNA binding dye structurally similar to Hoechst 33342 but with an excitation spectrum shifted toward the violet range, has also been used for SP analysis on flow cytometers equipped with violet laser diodes. In this chapter, we briefly review the history of this method and provide a detailed procedure. Critical parameters for good labeling, details on integrating simultaneous immunolabeling with DCV SP analysis, and proper data acquisition and analysis techniques are covered in detail.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
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. Goodell MA, Brose K, Paradis G, Connor AS, Mulligan RC (1996) Isolation and functional properties of murine hematopoetic stem cells that are replicating in vivo. J Exp Med 183:1797–1806

    Article  PubMed  CAS  Google Scholar 

  2. Challen GA, Little MH (2006) A side order of stem cells: the SP phenotype. Stem Cells 24:3–12

    Article  PubMed  Google Scholar 

  3. Weksberg DC, Chambers SM, Boles NC, Goodell MA (2008) CD150− side population cells represent a functionally distinct population of long-term hematopoietic stem cells. Blood 111:2444–2451

    Article  PubMed  CAS  Google Scholar 

  4. Dor FJ, Ramirez ML, Parmar K, Altman EL, Huang CA, Down JD, Cooper DK (2006) Primitive hematopoietic cell populations reside in the spleen: studies in the pig, baboon, and human. Exp Hematol 34:1573–1582

    Article  PubMed  CAS  Google Scholar 

  5. Kato K, Yoshimoto M, Kato K, Adachi S, Yamayoshi A et al (2007) Characterization of side-population cells in human normal endometrium. Hum Reprod 22:1214–1223

    Article  PubMed  CAS  Google Scholar 

  6. Summer R, Kotton DN, Sun X, Ma B, Fitzsimmons K, Fine A (2003) Side population cells and Bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol 285:L97–L104

    PubMed  CAS  Google Scholar 

  7. Hadnagy A, Gaboury L, Beaulieu R, Balicki D (2006) SP analysis may be used to identify cancer stem cell populations. Exp Cell Res 312:3701–3710

    Article  PubMed  CAS  Google Scholar 

  8. Brown MD, Gilmore PE, Hart CA, Samuel JD, Ramani VA, George NJ, Clarke NW (2007) Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations. Prostate 67:1384–1396

    Article  PubMed  Google Scholar 

  9. Fukuda K, Saikawa Y, Ohashi M, Kumagai K, Kitajima M, Okano H, Matsuzaki Y, Kitagawa Y (2009) Tumor initiating potential of side population cells in human gastric cancer. Int J Oncol 34:1201–1207

    PubMed  CAS  Google Scholar 

  10. Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, Barnard GF, Mori M (2006) Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 24:506–513

    Article  PubMed  CAS  Google Scholar 

  11. Ho MM, Ng AV, Lam S, Hung JY (2007) Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 67:4827–4833

    Article  PubMed  CAS  Google Scholar 

  12. Kim M, Turnquist H, Jackson J, Sgagias M, Yan Y, Gong M, Dean M, Sharp JG, Cowan K (2002) The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. Clin Cancer Res 8:22–28

    PubMed  CAS  Google Scholar 

  13. Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP (2002) Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci USA 99:12339–12344

    Article  PubMed  CAS  Google Scholar 

  14. Zhou S, Zong Y, Lu T, Sorrentino BP (2003) Hematopoietic cells from mice that are deficient in both Bcrp1/Abcg2 and Mdr1a/1b develop normally but are sensitized to mitoxantrone. Biotechniques 35:1248–1252

    PubMed  CAS  Google Scholar 

  15. Jonker JW, Freeman J, Bolscher E, Musters S, Alvi AJ, Titley I, Schinkel AH, Dale TC (2005) Contribution of the ABC transporters Bcrp1 and Mdr1a/1b to the side population phenotype in mammary gland and bone marrow of mice. Stem Cells 23:1059–1065

    Article  PubMed  CAS  Google Scholar 

  16. Morita Y (2006) Non-side-population hematopoietic stem cells in mouse bone marrow. Blood 108:2850–2856

    Article  PubMed  CAS  Google Scholar 

  17. Alt R, Wilhelm F, Pelz-Ackermann O, Egger D, Niederwieser D, Cross M (2009) ABCG2 expression is correlated neither to side population nor to hematopoietic progenitor function in human umbilical cord blood. Exp Hematol 37:294–301

    Article  PubMed  CAS  Google Scholar 

  18. Addla SK, Brown MD, Hart CA, Ramani VA, Clarke NW (2008) Characterization of the Hoechst 33342 side population from normal and malignant human renal epithelial cells. Am J Physiol Renal Physiol 295:F680–F687

    Article  PubMed  CAS  Google Scholar 

  19. Triel C, Vestergaard ME, Bolund L, Jensen TG, Jensen UB (2004) Side population cells in human and mouse epidermis lack stem cell characteristics. Exp Cell Res 295:79–90

    Article  PubMed  CAS  Google Scholar 

  20. Cabana R, Frolova EG, Thomas RA, Krishan A, Telford WG (2006) The minimal instrumentation requirements for Hoechst side population analysis: stem cell analysis on low-cost flow cytometry platforms. Stem Cells 24:2573–2581

    Article  PubMed  Google Scholar 

  21. Shapiro HM, Perlmutter NG (2001) Violet laser diodes as light sources for cytometry. Cytometry 44:133–136

    Article  PubMed  CAS  Google Scholar 

  22. Telford WG, Frolova EG (2004) Discrimination of Hoechst side population in mouse bone marrow with violet and near-UV laser diodes. Cytometry 57A:45–52

    Article  Google Scholar 

  23. Eaker SS, Hawley TS, Ramezani A, Hawley RG (2004) Detection and enrichment of hematopoetic stem cells by side population phenotype. In: Hawley TS, Hawley RG (eds) Methods molecular biology, vol 263, 2nd edn, Flow cytometry protocols. Humana, Totawa, NJ, pp 161–180

    Google Scholar 

  24. Simpson C, Pearce DJ, Bonnet D, Davies D (2006) Out of the blue: a comparison of Hoechst side population (SP) analysis of murine bone marrow using 325, 363 and 407 nm excitation sources. J Immunol Methods 310:171–181

    Article  PubMed  CAS  Google Scholar 

  25. Telford WG, Bradford J, Godfrey W, Robey RW, Bates SE (2007) Side population analysis using a violet-excited cell permeable DNA binding dye. Stem Cells 25:1029–1036

    Article  PubMed  CAS  Google Scholar 

  26. She JJ, Zhang PG, Wang ZM, Gan WM, Che XM (2008) Identification of side population cells from bladder cancer cells by DyeCycle Violet staining. Cancer Biol Ther 7: 1663–1668

    Article  PubMed  CAS  Google Scholar 

  27. Mathew G, Timm EA Jr, Sotomayor P, Godoy A, Montecinos VP, Smith GJ, Huss WJ (2009) ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate. Cell Cycle 8:1053–1061

    Article  PubMed  CAS  Google Scholar 

  28. Montanaro F, Liadaki K, Schienda J, Flint A, Gussoni E, Kunkel LM (2004) Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters. Exp Cell Res 298:144–154

    Article  PubMed  CAS  Google Scholar 

  29. Goodell MA (2005) Stem cell identification and sorting using the Hoechst 33342 side population (SP). In: Current protocols in cytometry. Wiley, New York, pp 9.18.1–9.18.11

    Google Scholar 

  30. Lin KK, Goodell MA (2006) Purification of hematopoietic stem cells using the side population. In: Methods in enzymology, vol 420. Elsevier, Inc., New York, pp 255–264

    Google Scholar 

  31. Petriz J (2007) Flow cytometry of the side population (SP). In: Current protocols in cytometry. Wiley, New York, pp 9.23.1–9.23.14

    Google Scholar 

  32. Telford WG (2010) Stem cell side population side population analysis and sorting using DyeCycle Violet. In: Robinson JP, Darzynkiewicz Z, Dobrucki J, Hoffman RA, Nolan JP, Orfao A, Rabinovitch PS (eds) Current protocols in cytometry, chap 9, Unit 9.30. Wiley, New York

    Google Scholar 

  33. Telford WG (2004) Analysis of UV-excited fluorochromes by flow cytometry using a near-UV laser diode. Cytometry 61A:9–17

    Article  Google Scholar 

  34. Zhao H, Traganos F, Dobrucki J, Wlodkowic D, Darzynkiewicz Z (2009) Induction of DNA damage response by the supravital probes of nucleic acids. Cytometry A 75: 510–519

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Experimental Transplantation and Immunology Branch, National Institutes of Health, Bethesda, MD, USA

    William G. Telford

Authors
  1. William G. Telford
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William G. Telford .

Editor information

Editors and Affiliations

  1. , Experimental Therapeutics, BC Cancer Agency, West 10th Ave. 675, Vancouver, V5Z 1L3, British Columbia, Canada

    Cheryl D. Helgason

  2. STEMCELL Technologies, Inc., W. Seventh Avenue 570, Vancouver, V5Z 1B3, British Columbia, Canada

    Cindy L. Miller

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Telford, W.G. (2013). Stem Cell Identification by DyeCycle Violet Side Population Analysis. In: Helgason, C., Miller, C. (eds) Basic Cell Culture Protocols. Methods in Molecular Biology, vol 946. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-128-8_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-128-8_11

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-127-1

  • Online ISBN: 978-1-62703-128-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation