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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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
Challen GA, Little MH (2006) A side order of stem cells: the SP phenotype. Stem Cells 24:3–12
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
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
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
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
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
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
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
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
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
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
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
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
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
Morita Y (2006) Non-side-population hematopoietic stem cells in mouse bone marrow. Blood 108:2850–2856
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
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
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
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
Shapiro HM, Perlmutter NG (2001) Violet laser diodes as light sources for cytometry. Cytometry 44:133–136
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
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
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
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
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
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
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
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
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
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
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
Telford WG (2004) Analysis of UV-excited fluorochromes by flow cytometry using a near-UV laser diode. Cytometry 61A:9–17
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
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights 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