Stem Cell Side Population Analysis and Sorting Using DyeCycle Violet

William G. Telford1

1 National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.30
DOI:  10.1002/0471142956.cy0930s51
Online Posting Date:  January, 2010
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Hoechst side population (SP) analysis has proven to be a valuable technique for identifying and sorting stem and early progenitor cells in a variety of tissues and species. In this method, the DNA binding dye Hoechst 33342 is loaded into the cell population of interest; stem cells preferentially exclude this dye, and these low‐fluorescence cells can be detected by flow cytometry. However, Hoechst SP analysis usually requires a flow cytometer equipped with an ultraviolet laser source for optimal performance. Unfortunately, ultraviolet lasers are expensive and are not common fixtures on flow cytometers. Violet laser diodes emitting in the 395‐ to 410‐nm range are less expensive and have become much more common on flow cytometers, but do not provide optimal excitation of Hoechst 33342. DyeCycle Violet is a cell‐permeable DNA binding dye with a chemical structure similar to Hoechst 33342, but with a longer excitation maximum. DyeCycle Violet can be substituted for Hoechst 33342 when performing side population analysis on a cytometer with a violet laser source. The procedure for DyeCycle Violet labeling for side population is described, as well as the limitations particular to this dye. Curr. Protoc. Cytom. 51:9.30.1‐9.30.9. © 2010 by John Wiley & Sons, Inc.

Keywords: stem cell; side population; Hoechst 33258; DyeCycle Violet

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • DMEM+ or HBSS+ (see reciperecipes)
  • Mouse bone marrow or human cord blood
  • Verapamil (see recipe)
  • Fumitremorgin C (see recipe)
  • DyeCycle Violet (10 mM DCV stock solution, Invitrogen)
  • 1 mg/ml propidium iodide (PI) stock solution
  • Surgical instruments
  • 50‐ml polypropylene tubes
  • 10‐ or 20‐ml syringes
  • 18‐ and 27‐G needles
  • 70‐µm mesh
  • 37°C water bath
  • Flow cytometer equipped with a violet laser diode, blue and red detection
  • Microsphere alignment beads
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Literature Cited

Literature Cited
   Challen, G.A. and Little, M.H. 2006. A side order of stem cells: The SP phenotype. Stem Cells 24:3‐12.
   Goodell, M.A., Brose, K., Paradis, G., Connor, A.S., and Mulligan, R.C. 1996. Isolation and functional properties of murine hematopoetic stem cells that are replicating in vivo. J. Exp. Med. 183:1797‐1806.
   Goodell, M.A. 2005. Stem cell identification and sorting using the Hoechst 33342 side population (SP). Curr. Protoc. Cytom. 34:9.18.1‐9.18.11.
   Hadnagy, A., Gaboury, L., Beaulieu, R., and Balicki, D. 2006. SP analysis may be used to identify cancer stem cell populations. Exp. Cell Res. 312:3701‐3710.
   Mathew, G., Timm, E.A. Jr., Sotomayor, P., Godoy, A., Montecinos, V.P., Smith, G.J., and Huss, W.J. 2009. ABCG2‐mediated DyeCycle Violet efflux defined side population in benign and malignant prostate. Cell Cycle 8:1053‐1061.
   She, J.J., Zhang, P.G., Wang, Z.M., Gan, W.M., and Che, X.M. 2008. Identification of side population cells from bladder cancer cells by DyeCycle Violet staining. Cancer Biol. Ther. 7:1663‐1668.
   Telford, W.G. and Frolova, E.G. 2004. Discrimination of Hoechst side population in mouse bone marrow with violet and near‐UV laser diodes. Cytometry 57:45‐52.
   Telford, W.G., Hawley, T.S., and Hawley, R.G. 2003. Analysis of violet‐excited fluorochromes by flow cytometry using a violet laser diode. Cytometry 54:48‐55.
   Telford, W.G., Kapoor, V., Jackson, J., Burgess, W., Buller, G., Hawley, T., and Hawley, R. 2006. Violet laser diodes in flow cytometry: An update. Cytometry 69:1153‐160.
   Telford, W.G., Bradford, J., Godfrey, W., Robey, R.W., and Bates, S.E. 2007. Side population analysis using a violet‐excited cell permeable DNA binding dye. Stem Cells 25:1029‐1036.
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