Visualisation of Signalling in Immune Cells
Visualisation of Signalling in Immune Cells
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DOI：
10.1007/978-1-60761-461-6_7
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发表时间：
2010-01-01
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影响因子：
--
通讯作者：
Ng, Tony
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文献类型：
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作者：
Carlin, Leo M.;Makrogianneli, Konstantina;Ng, Tony
目前，大量的方法被用于研究免疫系统。这些范围从活体动物实验和研究整个器官或细胞群的生化技术，到研究特定细胞相互作用动态的单细胞和分子技术。这是后一种方法，本章focuseson.The使用福斯特共振能量转移（FRET）技术探测蛋白质蛋白质的相互作用，参与受体信号传导到细胞骨架在完整的细胞，现在已经建立。各种FRET生物传感器可用于可视化几个关键的细胞过程，提供有关活动和关键信号分子位置的信息。作为本章中的一组具体示例，我们生成了原始Rho、Rac和Cdc42“Raichu”探针的变体，并改进了它们的荧光团组合，使其适用于FLIM。这些被用于许多测定中以确定T和NK细胞中的信号动力学。描述了如何使用这些探头的具体协议和技术说明。
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 Forster 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.