Protocol

Live Cell Imaging

Volume 591 of the series Methods in Molecular Biology pp 159-183

Date:

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

  • Hae Won SohnAffiliated withLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health
  • , Pavel TolarAffiliated withLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health
  • , Joseph BrzostowskiAffiliated withLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health
  • , Susan K. PierceAffiliated withLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health

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Abstract

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.

Key words

B lymphocyte fluorescence resonance energy transfer (FRET) total internal reflection fluorescence (TIRF) imaging B cell receptor (BCR) signaling planar lipid bilayer immune synapse