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A Live Cell Micro-imaging Technique to Examine Platelet Calcium Signaling Dynamics Under Blood Flow

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Platelets and Megakaryocytes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 788))

Abstract

The platelet is a specialized adhesive cell that plays a key role in thrombus formation under both physiological and pathological blood flow conditions. Platelet adhesion and activation are dynamic processes associated with rapid morphological and functional changes, with the earliest signaling events occurring over a subsecond time-scale. The relatively small size of platelets combined with the dynamic nature of platelet adhesion under blood flow means that the investigation of platelet signaling events requires techniques with both high spatial discrimination and rapid temporal resolution. Unraveling the complex signaling processes governing platelet adhesive function under conditions of hemodynamic shear stress has been a longstanding goal in platelet research and has been greatly influenced by the development and application of microimaging-based techniques. Advances in the area of epi-fluorescence and confocal-based platelet calcium (Ca2+) imaging have facilitated the in vitro and in vivo elucidation of the early signaling events regulating platelet adhesion and activation. These studies have identified distinct Ca2+ signaling mechanisms that serve to dynamically regulate activation of the major platelet integrin αIIbβ3 and associated adhesion and aggregation processes under flow. This chapter describes in detail a ratiometric calcium imaging protocol and associated troubleshooting procedures developed in our laboratory to examine live platelet Ca2+ signaling dynamics. This technique provides a method for high-resolution imaging of the Ca2+ dynamics underpinning platelet adhesion and thrombus formation under conditions of pathophysiological shear stress.

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Authors and Affiliations

  1. The Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia

    Warwick S. Nesbitt, Ian S. Harper, Simone M. Schoenwaelder, Yuping Yuan & Shaun P. Jackson

Authors
  1. Warwick S. Nesbitt
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  2. Ian S. Harper
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  3. Simone M. Schoenwaelder
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  4. Yuping Yuan
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  5. Shaun P. Jackson
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Corresponding author

Correspondence to Warwick S. Nesbitt .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, Inst. Cardiovascular & Metabolic Res., University of Reading, Reading, RG6 6UB, Berkshire, United Kingdom

    Jonathan M. Gibbins

  2. , Dept. Cell Physiology & Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN, United Kingdom

    Martyn P. Mahaut-Smith

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Nesbitt, W.S., Harper, I.S., Schoenwaelder, S.M., Yuan, Y., Jackson, S.P. (2012). A Live Cell Micro-imaging Technique to Examine Platelet Calcium Signaling Dynamics Under Blood Flow. 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_6

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  • DOI: https://doi.org/10.1007/978-1-61779-307-3_6

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-61779-306-6

  • Online ISBN: 978-1-61779-307-3

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