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Methods Mol Biol. 2010;591:159-83. doi: 10.1007/978-1-60761-404-3_10.

A method for analyzing protein-protein interactions in the plasma membrane of live B cells by fluorescence resonance energy transfer imaging as acquired by total internal reflection fluorescence microscopy.

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  • 1Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, USA.


For more than a decade, fluorescence resonance energy transfer (FRET) imaging methods have been developed to study dynamic interactions between molecules at the nanometer scale in live cells. Here, we describe a protocol to measure FRET by the acceptor-sensitized emission method as detected by total internal reflection fluorescence (TIRF) imaging to study the interaction of appropriately labeled plasma membrane-associated molecules that regulate the earliest stages of antigen-mediated signaling in live B lymphocytes. This protocol can be adapted and applied to many cell types where there is an interest in understanding signal transduction mechanisms in live cells.

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