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The Prospective Isolation of Viable, High Ploidy Megakaryocytes from Adult Murine Bone Marrow by Fluorescence Activated Cell Sorting

  • Shen Y. Heazlewood
  • Brenda Williams
  • Melonie J. Storan
  • Susan K. Nilsson
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1035)

Abstract

Mature megakaryocytes (MM) can be up to 65 μM in diameter and due to their size, viable and pure MM populations have been difficult to isolate in large numbers. Here in, we report a fluorescence activated cell sorting (FACS) method by which viable and pure populations of 8 N, 16 N, 32 N, and 64 N MM can be isolated from murine bone marrow (BM).

Key words

Megakaryocytes Ploidy Bone marrow Microenvironment Niche 

Notes

Acknowledgments

The authors thank Dani Cardozo for assistance with animal work, Michael Reitsma and Andrew Fryga for intellectual input and flow cytometric support. In addition, we also thank Kathryn Flanagan and Karen Clarke for flow cytometric support.

References

  1. 1.
    Shen Y, Nilsson SK (2012) Curr Opin Hematol 19(4):250–5Google Scholar
  2. 2.
    Heazlewood SY, Neaves RJ, Williams B, Haylock DN, Adams TE, Nilsson SK (in press) Stem Cell ResearchGoogle Scholar
  3. 3.
    Levine RF, Hazzard KC, Lamberg JD (1982) The significance of megakaryocyte size. Blood 60:1122–1131PubMedGoogle Scholar
  4. 4.
    Nakeff A, Maat B (1974) Separation of megakaryocytes from mouse bone marrow by velocity sedimentation. Blood 43:591–595PubMedGoogle Scholar
  5. 5.
    Levine RF, Fedorko ME (1976) Isolation of intact megakaryocytes from guinea pig femoral marrow. Successful harvest made possible with inhibitions of platelet aggregation; enrichment achieved with a two-step separation technique. J Cell Biol 69:159–172CrossRefPubMedGoogle Scholar
  6. 6.
    Radley JM, Haller CJ (1982) The demarcation membrane system of the megakaryocyte: a misnomer? Blood 60:213–219PubMedGoogle Scholar
  7. 7.
    Nakeff A, Valeriote F, Gray JW, Grabske RJ (1979) Application of flow cytometry and cell sorting to megakaryocytopoiesis. Blood 53:732–745PubMedGoogle Scholar
  8. 8.
    Jackson CW, Brown LK, Somerville BC, Lyles SA, Look AT (1984) Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions. Blood 63:768–778PubMedGoogle Scholar
  9. 9.
    Ishibashi T, Burstein SA (1985) Separation of murine megakaryocytes and their progenitors on continuous gradients of Percoll. J Cell Physiol 125:559–566CrossRefPubMedGoogle Scholar
  10. 10.
    Tanaka H, Ishida Y, Kaneko T, Matsumoto N (1989) Isolation of human megakaryocytes by immunomagnetic beads. Br J Haematol 73:18–22CrossRefPubMedGoogle Scholar
  11. 11.
    Kuter DJ, Gminski D, Rosenberg RD (1992) Botrocetin agglutination of rat megakaryocytes: a rapid method for megakaryocyte isolation. Exp Hematol 20:1085–1089PubMedGoogle Scholar
  12. 12.
    Hussein K (2011) Gene expression profiling in laser-microdissected bone marrow megakaryocytes. Methods Mol Biol 755:429–439CrossRefPubMedGoogle Scholar
  13. 13.
    Mazharian A (2012) Assessment of megakaryocyte migration and chemotaxis. Methods Mol Biol 788:275–288CrossRefPubMedGoogle Scholar
  14. 14.
    Tolhurst G, Carter RN, Miller N, Mahaut-Smith MP (2012) Purification of native bone marrow megakaryocytes for studies of gene expression. Methods Mol Biol 788:259–273CrossRefPubMedGoogle Scholar
  15. 15.
    Davies D (2007) Cell sorting by flow cytometry. In: Macey MG (ed) Flow cytometry: principles and applications, Humana Press, New Jersey, USA, pp 257–276.Google Scholar
  16. 16.
    Williams B, Nilsson SK (2009) Investigating the interactions between haemopoietic stem cells and their niche: methods for the analysis of stem cell homing and distribution within the marrow following transplantation. Methods Mol Biol 482:93–107CrossRefPubMedGoogle Scholar
  17. 17.
    Ebbe S, Boudreaux M (1998) Relationship of megakaryocyte ploidy with platelet number and size in cats, dogs, rabbits and mice. Comp Haematol Int 8:21–25CrossRefGoogle Scholar
  18. 18.
    Nilsson SK, Dooner MS, Tiarks CY, Weier HU, Quesenberry PJ (1997) Potential and distribution of transplanted hematopoietic stem cells in a nonablated mouse model. Blood 89:4013–4020PubMedGoogle Scholar
  19. 19.
    Haylock DN, Williams B, Johnston HM, Liu MC, Rutherford KE, Whitty GA et al (2007) Hemopoietic stem cells with higher hemopoietic potential reside at the bone marrow endosteum. Stem Cells 25:1062–1069CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Shen Y. Heazlewood
    • 1
  • Brenda Williams
    • 2
  • Melonie J. Storan
    • 2
  • Susan K. Nilsson
    • 2
    • 3
  1. 1.Materials Science and EngineeringCommonwealth Scientific and Industrial Research OrganizationMelbourneAustralia
  2. 2.Materials Science and EngineeringCommonwealth Scientific and Industrial Research OrganizationMelbourneAustralia
  3. 3.Department of Anatomy and Developmental BiologyMonash UniversityMelbourneAustralia

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