Abstract
Megakaryocytes constitute less than 1% of all marrow cells, therefore purification of these giant platelet precursor cells represents a challenge. We describe two methods to ultra-purify mature megakaryocytes from murine marrow for the purpose of extracting RNA suitable for studies of gene expression. In the first approach, unit velocity gradients are used to enrich for megakaryocytes, which are then selected by fluorescence-activated cell sorting based upon size and high surface expression of CD41. In the second method, individual megakaryocytes, identified by their distinct morphology, are extracted using glass suction pipettes. Despite the small numbers of cells that can be isolated via the latter technique, recent studies have demonstrated how this pure population can be used to detect mRNA transcripts encoding ion channels and other proteins in the native megakaryocyte.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wright, J. H. (1910) The histogenesis of the blood platelets Journal of Morphology 21, 263–278.
Wright, J. H. (1906) The origin and nature of the blood plates Boston Medical and Surgical Journal 154, 643–645.
Italiano, J. E., Jr. and Shivdasani, R. A. (2003) Megakaryocytes and beyond: the birth of platelets J Thromb. Haemost. 1, 1174–1182.
Shattil, S. J. and Leavitt, A. D. (2001) All in the family: primary megakaryocytes for studies of platelet αIIbβ3 signaling Thromb. Haemost. 86, 259–265.
Tolhurst, G., Vial, C., Leon, C., Gachet, C., Evans, R. J., and Mahaut-Smith, M. P. (2005) Interplay between P2Y1, P2Y12, and P2X1 receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling Blood 106, 1644–1651.
Mason, M. J. and Mahaut-Smith, M. P. (2004) Measurement and manipulation of intracellular Ca2+ in single platelets and megakaryocytes Methods Mol. Biol. 273, 251–276.
Mahaut-Smith, M. P. (2004) Patch-clamp recordings of electrophysiological events in the platelet and megakaryocyte Methods Mol. Biol. 273, 277–300.
Mason, M. J. and Mahaut-Smith, M. P. (2001) Voltage-dependent Ca2+ release in rat megakaryocytes requires functional IP3 receptors J Physiol 533, 175–183.
Tertyshnikova, S. and Fein, A. (1998) Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell Proc Natl Acad Sci USA 95, 1613–1617.
Monyer, H. and Jonas, P. (2010) in Single Channel Recording (Sakmann-B and Neher-H, Eds.).
Jonas, P., Racca, C., Sakmann, B., Seeburg, P. H., and Monyer, H. (1994) Differences in Ca2+ permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression Neuron 12, 1281–1289.
Lambolez, B., Audinat, E., Bochet, P., Crepel, F., and Rossier, J. (1992) AMPA receptor subunits expressed by single Purkinje cells Neuron 9, 247–258.
de Preter, K., Vandesompele, J., Heimann, P., Kockx, M. M., Van Gele, M., Hoebeeck, J., De Smet, E., Demarche, M., Laureys, G., Van Roy, N., De Paepe, A., and Speleman, F. (2003) Application of laser capture microdissection in genetic analysis of neuroblastoma and neuroblastoma precursor cells Cancer Lett. 197, 53–61.
Iyer, E. P. and Cox, D. N. (2010) Laser capture microdissection of Drosophila peripheral neurons J. Vis. Exp.
Carter, R. N., Tolhurst, G., Walmsley, G., Vizuete-Forster, M., Miller, N., and Mahaut-Smith, M. P. (2006) Molecular and electrophysiological characterization of transient receptor potential ion channels in the primary murine megakaryocyte J. Physiol 576, 151–162.
Raslova, H., Roy, L., Vourc’h, C., Le Couedic, J. P., Brison, O., Metivier, D., Feunteun, J., Kroemer, G., Debili, N., and Vainchenker, W. (2003) Megakaryocyte polyploidization is associated with a functional gene amplification Blood 101, 541–544.
Leven, R. M. (2004) Isolation of primary megakaryocytes and studies of proplatelet formation Methods Mol. Biol. 272, 281–292.
Acknowledgements
We thank Dr Stefan Amisten for comments on the manuscript. The techniques described in this chapter were developed as part of investigations of platelet and megakaryocyte ion channels, funded by the British Heart Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Tolhurst, G., Carter, R.N., Miller, N., Mahaut-Smith, M.P. (2012). Purification of Native Bone Marrow Megakaryocytes for Studies of Gene Expression. In: Gibbins, J., Mahaut-Smith, M. (eds) Platelets and Megakaryocytes. Methods in Molecular Biology, vol 788. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-307-3_18
Download citation
DOI: https://doi.org/10.1007/978-1-61779-307-3_18
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-61779-306-6
Online ISBN: 978-1-61779-307-3
eBook Packages: Springer Protocols