Through their B cell receptor (BCR), B cells recognise specific antigens that are internalized and directed to a specialized vesicular pathway. Precise coordination of the endosomal machinery is essential for processing the recognised antigens and triggering antibody responses.

A particular challenge is confronted in small and round cells, such as lymphocytes, where the reduced size of the cytoplasm and the resulting immediate vicinity of the plasma membrane generate major obstacles for imaging approaches.ī lymphocytes, or B cells, are a crucial part of the adaptive immune system, initiating the antibody responses against a vast repertoire of different antigens. Nevertheless, despite the improvements, imaging of vesicular pathways is challenged by the dynamic and heterogeneous nature of the vesicles and vesicular networks in all cell types. These improvements have increased our understanding of the location and behaviour of different vesicle populations, not possible to decipher otherwise using biochemical techniques 1, 2, 3, 4. In particular, in the last decade, improvements in fluorescence microscopy have aided the discovery of different vesicle subpopulations and transport machineries. Over the years, a general view of the vesicle pathways has been relatively well established. Intracellular membrane trafficking has not only critical housekeeping functions but also controls various cell-type-specific functions regulating, for instance, signalling cascades and cell fate. Our study demonstrates the potential of the SHIP assay for improving the imaging of internalized receptor/ligand complexes and establishes the compatibility of this assay with multiple imaging modalities, including live-cell imaging and super-resolution microscopy.

Here, we adopted the SHIP assay to study the trafficking of receptor/ligand complexes using B lymphocytes and B cell receptor-mediated antigen internalization as a model system. The specific hybridization internalisation probe (SHIP) assay, developed for flow cytometry studies, employs a ssDNA fluorescence internalisation probe and a complementary ssDNA quenching probe to unambiguously detect the internalized receptors/cargo.

However, distinguishing between plasma membrane-bound or internalised ligands remains a major challenge for the studies of cargo sorting to endosomal compartments, especially in small and round cells such as lymphocytes. Facilitated by the advancements in microscopy, our understanding of the complexity of intracellular vesicle traffic has dramatically increased in recent years.

For windows instal Super Warship