Visualisation of signalling in immune cells

Carlin, L. M. , Makrogianneli, K., Keppler, M., Fruhwirth, G. O. and Ng, T. (2010) Visualisation of signalling in immune cells. In: Marelli-Berg, F. M. and Nourshargh, S. (eds.) T-Cell Trafficking: Methods and Protocols. Series: Methods in molecular biology (616). Humana Press: New York, NY, pp. 97-113. ISBN 9781607614609 (doi:10.1007/978-1-60761-461-6_7)

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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.

Item Type:Book Sections
Additional Information: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).
Glasgow Author(s) Enlighten ID:Carlin, Dr Leo
Authors: Carlin, L. M., Makrogianneli, K., Keppler, M., Fruhwirth, G. O., and Ng, T.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Publisher:Humana Press
Published Online:15 February 2010

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