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.
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.
Sohn
Hae Won
HW
Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, USA.
Tolar
Pavel
P
Brzostowski
Joseph
J
Pierce
Susan K
SK
D000818:Animals; D000938:Antigen-Presenting Cells / Q000378:metabolism; D000941:Antigens / Q000378:metabolism; D001402:B-Lymphocytes / Q000166:cytology* / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer; D007091:Image Processing, Computer-Assisted; D008051:Lipid Bilayers / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D019061:src-Family Kinases / Q000378:metabolism
D000941:Antigens; D008051:Lipid Bilayers; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases