Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP)

Margaret A. Goodell1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.18
DOI:  10.1002/0471142956.cy0918s34
Online Posting Date:  November, 2005
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


This unit describes the use of Hoechst 33342 to identify and purify murine hematopoietic stem cells, the so‐called side population. Three properties of the dye contribute to the ability to distinguish stem cells in this way. Hoechst is a DNA‐binding dye. It has at least two binding modes that result in different spectral properties, allowing resolution of multiple populations by viewing fluorescence at two wavelengths simultaneously. The ability to discriminate SP cells is based on the differential efflux of the dye by a multi‐drug‐like transporter. Particular attention is given to the critical aspects of Hoechst concentration, cell concentration, staining time, and staining temperature.

PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Staining and Analysis of Stem Cell Side Population in Murine Bone Marrow
  • Support Protocol 1: Preparation of Mouse Bone Marrow
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Staining and Analysis of Stem Cell Side Population in Murine Bone Marrow

  • DMEM+ (see recipe)
  • Mouse bone marrow cells prepared according to protocol 2, or bone marrow cells from another species, or cells from non‐bone marrow tissue
  • 1 mg/ml Hoechst 33342 (see recipe)
  • Hanks buffered salt solution+ (HBSS+, see recipe), on ice
  • 200 µg/ml propidium iodide in PBS (see recipe; optional)
  • 5 mM verapamil in 95% ethanol (optional; Sigma)
  • Circulating water bath at exactly 37°C
  • 250‐ml or 50‐ml polypropylene centrifuge tubes (Corning)
  • Flow cytometer with UV laser (usually high‐power argon tuned to 351‐ to 364‐nm excitation) and standard 488‐nm laser
  • 450/20 band‐pass (BP) filter
  • 675‐nm edge filter long‐pass (EFLP; Omega Optical)
  • 610‐nm dichroic mirror short‐pass (DMSP) filter
  • Additional reagents and equipment for counting cells ( appendix 3A)

Support Protocol 1: Preparation of Mouse Bone Marrow

  • Mouse (the author uses C57Bl/6, but other strains will work)
  • HBSS+ (see recipe)
  • 70% ethanol in spray bottle
  • Rugged and delicate scissors
  • Small forceps
  • 10‐cm petri dishes
  • 10‐ml syringes
  • 18‐ and 27‐G needles
  • 50‐ml tubes
  • 70‐µm filter or mesh
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Chen, A.Y., Yu, C., Gatto, B., and Liu, L.F. 1993. DNA minor groove‐binding ligands: A different class of mammalian DNA topoisomerase I inhibitors. Proc. Natl. Acad. Sci. U.S.A. 90:8131‐8135.
   Goodell, M.A., Brose, K., Paradis, G., Conner, A.S., and Mulligan, R.C. 1996. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183:1797‐1806.
   Goodell, M.A., Rosenzweig, M., Kim, H., Marks, D.F., DeMaria, M., Paradis, G., Grupp, S.A., Sieff, C.A., Mulligan, R.C., and Johnson, R.P. 1997. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat. Med. 3:1337‐1345.
   Gussoni, E., Soneoka, Y., Strickland, C.D., Buzney, E.A., Khan, M.K., Flint, A.F., Kunkel, L.M., and Mulligan, R.C. 1999. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401:390‐394.
   Jackson, K.A., Mi, T., and Goodell, M.A. 1999. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc. Natl. Acad. Sci. U.S.A. 96:14482‐14486.
   Jordan, C.T., Astle, C.M., Zawadzki, J., Mackarehtschian, K., Lemischka, I.R., and Harrison, D.E. 1995. Long‐term repopulating abilities of enriched fetal liver stem cells measured by competitive repopulation. Exp. Hematol. 23:1011‐1015.
   Loken, M.R. 1980. Separation of viable T and B lymphocytes using a cytochemical stain, Hoechst 33342. J. Histochem. Cytochem. 28:36‐39.
   Morrison, S. and Weissman, I. 1994. The long‐term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1:661‐673.
   Pallavicini, M.G., Summers, L.J., Dean, P.N., and Gray, J.W. 1985. Enrichment of murine hemopoietic clonogenic cells by multivariate analyses and sorting. Exp. Hematol. 13:1173‐1181.
   Sandhu, L.C., Warters, R.L., and Dethlefsen, L.A. 1985. Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA. Cytometry 6:191‐194.
   Schinkel, A., Mayer, U., Wagenaar, E., Mol, C., van Deemter, L., Smit, J., van der Valk, M., Voordouw, A., Spits, H., van Tellingen, O., Zijlmans, J., Fibbe, W., and Borst, P. 1997. Normal viability and altered pharmacokinetics in mice lacking mdr1‐type (drug‐transporting) P‐glycoproteins. Proc. Natl. Acad. Sci. U.S.A. 94:4028‐4033.
   Spangrude, G.J. and Johnson, G.R. 1990. Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc. Natl. Acad. Sci. U.S.A. 87:7433‐7437.
   Spangrude, G.J., Heimfeld, S., and Weissman, I.L. 1988. Purification and characterization of mouse hematopoietic stem cells. Science 241:58‐62.
   Sriram, M., van der Marel, G.A., Roelen, H.L., van Boom, J.H., and Wang, A.H. 1992. Conformation of B‐DNA containing O6‐ethyl‐G‐C base pairs stabilized by minor groove binding drugs: Molecular structure of d(CGC[e6G]AATTCGCG complexed with Hoechst 33258 or Hoechst 33342. EMBO J. 11:225‐232.
   Steen, H.B. and Stokke, T. 1986. Fluorescence spectra of cells stained with a DNA‐specific dye, measured by flow cytometry. Cytometry 7:104‐106.
   Stokke, T. and Steen, H.B. 1986. Binding of Hoechst 33258 to chromatin in situ. Cytometry 7:227‐234.
   Storms, R.W., Goodell, M.A., Fisher, A., Mulligan, R.C., and Smith, C. 2000. Hoechst dye efflux reveals a novel CD7(+)CD34(−) lymphoid progenitor in human umbilical cord blood. Blood 96:2125‐2133.
   Uchida, N. and Weissman, I.L. 1992. Searching for hematopoietic stem cells: Evidence that Thy‐1.1lo Lin− Sca‐1+ cells are the only stem cells in C57BL/Ka‐Thy‐1.1 bone marrow. J. Exp. Med. 175:175‐184.
   Watson, J.V., Nakeff, A., Chambers, S.H., and Smith, P.J. 1985. Flow cytometric fluorescence emission spectrum analysis of Hoechst‐33342‐stained DNA in chicken thymocytes. Cytometry 6:310‐315.
   Wolf, N.S., Kone, A., Priestley, G.V., and Bartelmez, S.H. 1993. In vivo and in vitro characterization of long‐term repopulating primitive hematopoietic cells isolated by sequenctional Hoechst 33342‐rhodamine 123 FACS selection. Exp. Hematol. 21:614‐622.
   Zhou, S., Schuetz, J.D., Bunting, K.D., Colapietro, A.M., Sampath, J., Morris, J.J., Lagutina, I., Grosveld, G.C., Osawa, M., Nakauchi, H., and Sorrentino, B.P. 2001. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side‐population phenotype. Nat. Med. 7:1028‐1034.
PDF or HTML at Wiley Online Library