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Visualisation of Signalling in Immune Cells

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T-Cell Trafficking

Part of the book series: Methods in Molecular Biology ((MIMB,volume 616))

Abstract

Currently, a great number of approaches are employed in investigation of the immune system. These range from experiments in live animals and biochemical techniques to investigate whole organs or cell populations down to single cell and molecular techniques to look at dynamics in specific cell–cell interactions. It is the latter approach that this chapter focusses on. The use of Förster resonance energy transfer (FRET) techniques to probe protein–protein interactions that are involved in receptor signalling to the cytoskeleton in intact cells is now well established. Various FRET biosensors are available to visualise several critical cell processes, giving information about activity and the location of key signalling molecules. As a specific set of examples in this chapter, we have generated variants of the original Rho, Rac and Cdc42 “Raichu” probes and improved their fluorophore combination to make them suitable for FLIM. These were employed in a number of assays to determine signal dynamics in T and NK cells. Specific protocols of how to use these probes and technical notes are described.

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Acknowledgments

Konstantina Makrogianneli was supported by a joint research studentship from MRC/King’s College London. Leo Carlin is supported by a UK EPSRC grant (EP/C546105/1). G. Fruhwirth is supported by the King’s College London and University College London Comprehensive Cancer Imaging Center CR-UK & EPSRC in association with MRC and DOH (England) C1519/A10331 M. Keppler and T. Ng are supported by an endowment fund from the Dimbleby Cancer Care to King’s College London. The multi-photon FLIM system was built with support from both the Medical Research Council Co-Operative Group grant (G0100152 ID 56891) and a UK Research Councils Basic Technology Research Programme grant (GR/R87901/01).

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Authors and Affiliations

  1. Cancer Studies Division/Randall Division of Cellular and Molecular Biophysics, Richard Dimbleby Department of Cancer Research, Guy’s Medical School Campus, King’s College London, London, UK

    Leo M. Carlin, Konstantina Makrogianneli, Melanie Keppler, Gilbert O. Fruhwirth & Tony Ng

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  1. Leo M. Carlin
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  2. Konstantina Makrogianneli
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  3. Melanie Keppler
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  4. Gilbert O. Fruhwirth
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  5. Tony Ng
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Editors and Affiliations

  1. Hammersmith Hospital, Dept. Immunology, Imperial College London, Du Cane Road, London, W12 0NN, United Kingdom

    Federica M. Marelli-Berg

  2. School of Medicine and Dentistry, William Harvey Research Inst., Queen Mary, University of London, Charterhouse Square, London, EC1M 6BQ, United Kingdom

    Sussan Nourshargh

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Carlin, L.M., Makrogianneli, K., Keppler, M., Fruhwirth, G.O., Ng, T. (2010). Visualisation of Signalling in Immune Cells. In: Marelli-Berg, F., Nourshargh, S. (eds) T-Cell Trafficking. Methods in Molecular Biology, vol 616. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-461-6_7

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  • DOI: https://doi.org/10.1007/978-1-60761-461-6_7

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-460-9

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