Stem Cell Identification by DyeCycle Violet Side Population Analysis

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


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.

Key words

Stem cell Progenitor Flow cytometry Side population Hoechst 33342 DyeCycle Violet 


  1. 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 183:1797–1806PubMedCrossRefGoogle Scholar
  2. 2.
    Challen GA, Little MH (2006) A side order of stem cells: the SP phenotype. Stem Cells 24:3–12PubMedCrossRefGoogle Scholar
  3. 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 111:2444–2451PubMedCrossRefGoogle Scholar
  4. 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 34:1573–1582PubMedCrossRefGoogle Scholar
  5. 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 22:1214–1223PubMedCrossRefGoogle Scholar
  6. 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–L104PubMedGoogle Scholar
  7. 7.
    Hadnagy A, Gaboury L, Beaulieu R, Balicki D (2006) SP analysis may be used to identify cancer stem cell populations. Exp Cell Res 312:3701–3710PubMedCrossRefGoogle Scholar
  8. 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 67:1384–1396PubMedCrossRefGoogle Scholar
  9. 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 34:1201–1207PubMedGoogle Scholar
  10. 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 24:506–513PubMedCrossRefGoogle Scholar
  11. 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 67:4827–4833PubMedCrossRefGoogle Scholar
  12. 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 8:22–28PubMedGoogle Scholar
  13. 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 99:12339–12344PubMedCrossRefGoogle Scholar
  14. 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 35:1248–1252PubMedGoogle Scholar
  15. 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 23:1059–1065PubMedCrossRefGoogle Scholar
  16. 16.
    Morita Y (2006) Non-side-population hematopoietic stem cells in mouse bone marrow. Blood 108:2850–2856PubMedCrossRefGoogle Scholar
  17. 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 37:294–301PubMedCrossRefGoogle Scholar
  18. 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–F687PubMedCrossRefGoogle Scholar
  19. 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 295:79–90PubMedCrossRefGoogle Scholar
  20. 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 24:2573–2581PubMedCrossRefGoogle Scholar
  21. 21.
    Shapiro HM, Perlmutter NG (2001) Violet laser diodes as light sources for cytometry. Cytometry 44:133–136PubMedCrossRefGoogle Scholar
  22. 22.
    Telford WG, Frolova EG (2004) Discrimination of Hoechst side population in mouse bone marrow with violet and near-UV laser diodes. Cytometry 57A:45–52CrossRefGoogle Scholar
  23. 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 161–180Google Scholar
  24. 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 310:171–181PubMedCrossRefGoogle Scholar
  25. 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 25:1029–1036PubMedCrossRefGoogle Scholar
  26. 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: 1663–1668PubMedCrossRefGoogle Scholar
  27. 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 8:1053–1061PubMedCrossRefGoogle Scholar
  28. 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 298:144–154PubMedCrossRefGoogle Scholar
  29. 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 9.18.1–9.18.11Google Scholar
  30. 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 255–264Google Scholar
  31. 31.
    Petriz J (2007) Flow cytometry of the side population (SP). In: Current protocols in cytometry. Wiley, New York, pp 9.23.1–9.23.14Google Scholar
  32. 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 YorkGoogle Scholar
  33. 33.
    Telford WG (2004) Analysis of UV-excited fluorochromes by flow cytometry using a near-UV laser diode. Cytometry 61A:9–17CrossRefGoogle Scholar
  34. 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: 510–519PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Experimental Transplantation and Immunology BranchNational Institutes of HealthBethesdaUSA

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