Binding of antigen to the B cell receptor (BCR) triggers both BCR signaling and endocytosis simultaneously. BCR signaling pathways and their regulation have been studied extensively by both biochemical methods and flow cytometry, resulting in a comprehensive understanding of the temporal dynamics of the signaling enzymes and effector proteins. However, spatial regulation of these signaling pathways in subcellular pathways is relatively poorly understood. Here, we describe a method to study the spatio-temporal distribution of phosphorylated-kinases in antigen-activated B cells by confocal microscopy. This method can also be applied to other cell types where it is of interest to understand the spatial distribution of signaling enzymes and their effector proteins.
Keywords: B cell receptors; B cells; BCR signaling; Confocal microscopy; Endocytosis.