Visualisation of signalling in immune cells

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