Skip to main content
SpringerLink
Log in

Hematopoietic Stem Cell Characterization by Hoechst 33342 and Rhodamine 123 Staining

  • Protocol
Flow Cytometry Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 263))

Abstract

A dual-dye efflux strategy utilizing the supravital dyes Hoechst 33342 (Ho) and rhodamine 123 (Rh123) is described and illustrated for the detection and analysis of hematopoietic stem cells in murine bone marrow. Mononuclear cells from bone marrow cell suspensions were incubated in a cocktail of Rh123 plus Ho, and both dyes were effluxed by two 15-min incubations in dye-free buffer prior to sorting. Compared to our original prototype method in which Rh123, but not Ho, was effluxed, this dual-dye efflux protocol more rapidly and efficiently resolves the most primitive Hodull/Rhdull hematopoietic stem cells. Moreover, under conditions of optimal dual-dye uptake and efflux, Hodull/Rhdull cells map to the subfraction of side population (SP) cells with the highest efflux of Ho, which were previously demonstrated to possess the highest hematopoietic stem cell activity.

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight 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. Bertoncello, I., Hodgson, G. S., and Bradley, T R. (1985) Multiparameter analysis of transplantable hemopoietic stem cells. I. The separation and enrichment of stem cells homing to marrow and spleen on the basis of Rhodamine 123 fluorescence. Exp. Hematol. 13, 999–1006.

    PubMed  CAS  Google Scholar 

  2. Bertoncello, I., Hodgson, G S., and Bradley, T R. (1988) Multiparameter analysis of transplantable hemopoietic stem cells. II. Stem cells of long-term bone marrow reconstituted recipients. Exp. Hematol. 16, 245–249.

    PubMed  CAS  Google Scholar 

  3. Bertoncello, I., Bradley, T R., Hodgson, G S., and Dunlop, J. M. (1991) The resolution, enrichment, and organisation of normal bone marrow high proliferative potential colony-forming cell subsets on the basis of rhodamine 123 fluorescence. Exp. Hematol. 19, 174–178.

    PubMed  CAS  Google Scholar 

  4. Visser, J. W., Bol, S. J., and van den Engh, G (1981) Characterization and enrichment of murine hemopoietic stem cells by fluorescence activated cell sorting. Exp. Hematol. 9, 644–655.

    PubMed  CAS  Google Scholar 

  5. Baines, P. and Visser, J. W. (1983) Analysis and separation of murine bone marrow stem cells by H33342 fluorescence-activated cell sorting. Exp. Hematol. 11, 701–708.

    PubMed  CAS  Google Scholar 

  6. Ploemacher, R. E. and Brons, N. H. (1988) Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12 and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention. J. Cell. Physiol. 136, 531–536.

    Article  PubMed  CAS  Google Scholar 

  7. 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.

    PubMed  CAS  Google Scholar 

  8. Neben, S., Redfearn, W. J., Parra, M., Brecher, G., and Pallavicini, M. (1991) Short-and long-term repopulation of lethally irradiated mice by bone marrow stem cells enriched on the basis of light scatter and Hoechst 33342 fluorescence. Exp. Hematol. 19, 958–967.

    PubMed  CAS  Google Scholar 

  9. 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.

    Article  PubMed  CAS  Google Scholar 

  10. Goodell, M. A., Rosenzweig, M., Kim, H., et al. (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.

    Article  PubMed  CAS  Google Scholar 

  11. 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 sequential Hoechst 33342-rhodamine 123 FACS selection. Exp. Hematol. 21, 614–622.

    PubMed  CAS  Google Scholar 

  12. Sitnicka, E., Ruscetti, F. W., Priestley, G. V., Wolf, N. S., and Bartelmez, S. H. (1996) Transforming growth factor beta 1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells. Blood 88, 82–88.

    PubMed  CAS  Google Scholar 

  13. Bradford, G. B., Williams, B., Rossi, R., and Bertoncello, I. (1997) Quiescence, cycling and turnover in the primitive hematopoietic stem cell compartment. Exp. Hematol. 25, 445–453.

    PubMed  CAS  Google Scholar 

  14. Nilsson, S., Dooner, M., Tiarks, C, Heinz-Ulrich, W., and Quesenberry, P. J. (1997) Potential and distribution of transplanted hematopoietic stem cells in a nonablated mouse model. Blood 89, 4013–4020.

    PubMed  CAS  Google Scholar 

  15. Reddy, G. P., Tiarks, C. Y., Pang, L., Wuu, J., Hsieh, C. C, and Quesenberry, P. J. (1997) Cell cycle analysis and synchronization of pluripotent hematopoietic progenitor stem cells. Blood 90, 2293–2299.

    PubMed  CAS  Google Scholar 

  16. Micallef, S, Ramsay, R., Williams, B., Rossi, R., Mucenski, M., and Bertoncello, I. (2000) The role of c-myb and BCL-2 in hematopoietic stem cell regulation. Exp. Hematol. 28(Suppl. 1), 68.

    Google Scholar 

  17. Reddy, G. P. V., McAuliffe, C. I., Pang, L., Quesenberry, P. J., and Bertoncello, I. (2002) Cytokine receptor repertoire and cytokine responsiveness of Hodull/Rhdull stem cells with differing potentials for G1/S phase progression. Exp. Hematol. 30, 792–800.

    Article  PubMed  CAS  Google Scholar 

  18. Spangrude, G. J. and Johnson, G. R. (1990) Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 87, 7433–7437.

    Article  PubMed  CAS  Google Scholar 

  19. Li, C. L. and Johnson, G. R. (1995) Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization. Blood 85, 1472–1479.

    PubMed  CAS  Google Scholar 

  20. Uchida, N., Combs, J., Chen, S., Zanjani, E., Hoffman, R., and Tsukamoto, A. (1996) Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities. Blood 88, 1297–1305.

    PubMed  CAS  Google Scholar 

  21. Kim, M., Cooper, D. D., Hayes, S. F., and Spangrude, G. J. (1998) Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux. Blood 91, 4106–4117.

    PubMed  CAS  Google Scholar 

  22. Kim, M., Moon, H. B., and Spangrude, G. J. (2003) Major age-related changes of mouse hematopoietic stem/progenitor cells. Ann. NY Acad. Sci. 996, 195–208.

    Article  PubMed  Google Scholar 

  23. Zijlmans, J. M., Visser, J. W., Kleiverda, K., Kluin, P. M., Willemze, R., and Fibbe, W. E. (1995) Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-repopulating ability. Proc. Natl. Acad. Sci. USA 92, 8901–8905.

    Article  PubMed  CAS  Google Scholar 

  24. Visser, J. W. and de Vries, P. (1988) Isolation of spleen-colony forming cells (CFU-s) using wheat germ agglutinin and rhodamine 123 labeling. Blood Cells 14, 369–384.

    PubMed  CAS  Google Scholar 

  25. Huttmann, A., Liu, S. L., Boyd, A. W., and Li, C. L. (2001) Functional heterogeneity within rhodamine123(lo) Hoechst33342(lo/sp) primitive hemopoietic stem cells revealed by pyronin Y. Exp. Hematol. 29, 1109–1116.

    Article  PubMed  CAS  Google Scholar 

  26. Srour, E. F. and Jordan, C. T. (2002) Isolation and characterization of primitive hematopoietic cells based on their position in the cell cycle, in Hematopoietic Stem Cell Protocols (Klug, C. A. and Jordan, C. T., eds.), Humana Press, Totowa, NJ, pp. 93–111.

    Google Scholar 

  27. Hellman, S., Botnick, L. E., Hannon, E. C, and Vigneulle, R. M. (1978) Proliferative capacity of murine hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 75, 490–494.

    Article  PubMed  CAS  Google Scholar 

  28. Lajtha, L. G (1979) Stem cell concepts. Differentiation 14, 23–34.

    Article  PubMed  CAS  Google Scholar 

  29. Hodgson, G. S., Bradley, T. R., and Radley, J. M. (1982) The organization of hemopoietic tissue as inferred from the effects of 5-fluorouracil. Exp. Hematol. 10, 26–35.

    PubMed  CAS  Google Scholar 

  30. Bunting, K. D. (2002) ABC transporters as phenotypic markers and functional regulators of stem cells. Stem Cells 20, 11–20.

    Article  PubMed  CAS  Google Scholar 

  31. Schinkel, A. H., Smit, J. J., van Tellingen, O., et al. (1994) Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell 77, 491–502.

    Article  PubMed  CAS  Google Scholar 

  32. Schinkel, A. H, Mayer, U., Wagenaar, E., et al. (1997) Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins. Proc. Natl. Acad. Sci. USA 94, 4028–4033.

    Article  PubMed  CAS  Google Scholar 

  33. Scharenberg, C. W., Harkey, M. A., and Torok-Storb, B. (2002) The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 99, 507–512.

    Article  PubMed  CAS  Google Scholar 

  34. Zhou, S., Schuetz, J. D., Bunting, K. D., et al. (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.

    Article  PubMed  CAS  Google Scholar 

  35. Zhou, S., Morris, J. J., Barnes, Y., Lan, L., Schuetz, J. D., and Sorrentino, B. P. (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, 12,339–12,344.

    Article  PubMed  CAS  Google Scholar 

  36. Johnson, L. V., Walsh, M. L., and Chen, L. B. (1980) Localization of mitochondria in living cells with rhodamine 123. Proc. Natl. Acad. Sci. USA 77, 990–994.

    Article  PubMed  CAS  Google Scholar 

  37. Johnson, L. V., Walsh, M. L., Bockus, B. J., and Chen, L. B. (1981) Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J. Cell Biol. 88, 526–535.

    Article  PubMed  CAS  Google Scholar 

  38. Darzynkiewicz, Z., Staiano-Coico, L., and Melamed, M. R. (1981) Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc. Natl. Acad. Sci. USA 78, 2383–2387.

    Article  PubMed  CAS  Google Scholar 

  39. Latt, S. A. and Stetten, G. (1976) Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis. J. Histochem. Cytochem. 24, 24–33.

    PubMed  CAS  Google Scholar 

  40. Lalande, M. E. and Miller, R. G. (1979) Fluorescence flow analysis of lymphocyte activation using Hoechst 33342 dye. J. Histochem. Cytochem. 27, 394–397.

    PubMed  CAS  Google Scholar 

  41. Arndt-Jovin, D. J. and Jovin, T. M. (1977) Analysis and sorting of living cells according to deoxyribonucleic acid content. J. Histochem. Cytochem. 25, 585–589.

    PubMed  CAS  Google Scholar 

  42. 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.

    Article  PubMed  CAS  Google Scholar 

  43. Smith, P. J., Nakeff, A., and Watson, J. V. (1985) Flow-cytometric detection of changes in the fluorescence emission spectrum of a vital DNA-specific dye in human tumour cells. Exp. Cell Res. 159, 37–46.

    Article  PubMed  CAS  Google Scholar 

  44. Smith, P. J., Morgan, S. A., and Watson, J. V. (1991) Detection of multidrug resistance and quantification of responses of human tumour cells to cytotoxic agents using flow cytometric spectral shift analysis of Hoechst 33342-DNA fluorescence. Cancer Chemother. Pharmacol. 27, 445–450.

    Article  PubMed  CAS  Google Scholar 

  45. Ratajczak, M. Z., Pletcher, C. H., Marlicz, W., et al. (1998) CD34+, kit+, rhodamine123(low) phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells. Leukemia 12, 942–950.

    Article  PubMed  CAS  Google Scholar 

  46. Leemhuis, T., Yoder, M. C, Grigsby, S., Aguero, B., Eder, P., and Srour, E. F (1996) Isolation of primitive human bone marrow hematopoietic progenitor cells using Hoechst 33342 and Rhodamine 123. Exp. Hematol. 24, 1215–1224.

    PubMed  CAS  Google Scholar 

  47. Szilvassy, S. J. and Cory, S. (1993) Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil-treated murine marrow. Blood 81, 2310–2320.

    PubMed  CAS  Google Scholar 

  48. Borth, N., Kral, G, and Katinger, H. (1993) Rhodamine 123 fluorescence of immortal hybridoma cell lines as a function of glucose concentration. Cytometry 14, 70–73.

    Article  PubMed  CAS  Google Scholar 

  49. Lambert, J. F., Carlson, J. E., Colvin, G. A., and Quesenberry, P. J. (2000) Evaluation of mouse whole body bone marrow cellularity and distribution of hematopoietic progenitors. Exp. Hematol. 28, 1493.

    Article  Google Scholar 

  50. Nilsson, S. K., Johnston, H. M., and Coverdale, J. A. (2001) Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches. Blood 97, 2293–2299.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Stem Cell Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia

    Ivan Bertoncello & Brenda Williams

Authors
  1. Ivan Bertoncello
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brenda Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. American Red Cross, Rockville, MD

    Teresa S. Hawley  & Robert G. Hawley  & 

  2. The George Washington University, Washington, DC

    Robert G. Hawley

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc.

About this protocol

Cite this protocol

Bertoncello, I., Williams, B. (2004). Hematopoietic Stem Cell Characterization by Hoechst 33342 and Rhodamine 123 Staining. In: Hawley, T.S., Hawley, R.G. (eds) Flow Cytometry Protocols. Methods in Molecular Biology™, vol 263. Humana Press. https://doi.org/10.1385/1-59259-773-4:181

Download citation

  • DOI: https://doi.org/10.1385/1-59259-773-4:181

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-234-6

  • Online ISBN: 978-1-59259-773-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation