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Calcium Signaling Protocols pp 175–192Cite as

Single-Cell Imaging Techniques for the Real-Time Detection of IP3 in Live Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 937))

Abstract

Inositol 1,4,5-trisphosphate (IP3) is a ubiquitous second messenger, derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by enzymes of the phospholipase C (PLC) family. Binding of IP3 to its cognate receptor in the endoplasmic reticulum membrane leads to release of Ca2+ into the cytoplasm, which is involved in the regulation of an array of cellular functions. Traditional techniques for the detection of IP3 have required the extraction of a large number of cells, with limitations in the time resolution of changes in IP3 and an inability to obtain detailed information on the dynamics of this second messenger in single cells. Recent progress in this field has led to the development of a number of genetically encoded fluorescent biosensors, which upon recombinant expression are able selectively to detect real-time changes in IP3 in single live cells. In this chapter, I detail protocols for the expression, visualization (by confocol or fluorescence microscopy), and interpretation of data obtained with such biosensors expressed in mammalian cells.

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Acknowledgements

I gratefully acknowledge Prof. Tobias Meyer (Stanford University, USA) for making the eGFP-PH biosensor available to us and Prof. Katsuhiko Mikoshiba (RIKEN, Japan) for providing us with the IRIS-1 biosensor. I also thank Prof. John Challiss (University of Leicester, UK) for his critical reading of the manuscript.

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Authors and Affiliations

  1. Department of Cardiovascular Sciences, Division of Anaesthesia, Critical Care, and Pain Management, Leicester Royal Infirmary, University of Leicester, Leicester, UK

    Carl P. Nelson

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  1. Carl P. Nelson
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Correspondence to Carl P. Nelson .

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Editors and Affiliations

  1. , Department of Cardiovascular Sciences, Leicester Royal Infirmary, Infirmary Square, Leicester, LE1 5WW, Leicestershire, United Kingdom

    David G. Lambert

  2. , Department of Cardiovascular Sciences, Leicester Royal Infirmary, Infirmary Square, Leicester, LE1 5WW, Leicestershire, United Kingdom

    Richard D. Rainbow

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Nelson, C.P. (2013). Single-Cell Imaging Techniques for the Real-Time Detection of IP3 in Live Cells. In: Lambert, D., Rainbow, R. (eds) Calcium Signaling Protocols. Methods in Molecular Biology, vol 937. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-086-1_10

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  • DOI: https://doi.org/10.1007/978-1-62703-086-1_10

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-085-4

  • Online ISBN: 978-1-62703-086-1

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