Abstract
Facilitated by the advancements in microscopy, our understanding of the complexity of intracellular vesicle traffic has dramatically increased in recent years. 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. 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. 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. 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.
Highlights
Intracellular membrane trafficking has critical housekeeping functions and controls various celltype-specific functions regulating, for instance, signalling cascades and cell fate
With the aim to find a solution for these obstacles, we set up the specific hybridization internalization probe (SHIP) assay to image internalized antigen in B cells
The SHIP assay requires two different oligos: a fluorescent single-stranded DNA (ssDNA) internalisation probe, that is conjugated to the desired ligand or antibody (FIP; 5′ fluorophore-TCAGTTCAGGACCCTCGGCT-N3 3′), and a complementary ssDNA-containing quenching probe (QP; 5′ AGCCGAGGGTCCTGAACTGA-Quencher 3′) (Fig. 1B)
Summary
Intracellular membrane trafficking has critical housekeeping functions and controls various celltype-specific functions regulating, for instance, signalling cascades and cell fate. Precise coordination of the endosomal machinery is essential for processing the recognised antigens and triggering antibody responses Through their B cell receptor (BCR), B cells recognise specific antigens that are internalized and directed to a specialized vesicular pathway. In 2013, Liu and Johnston developed an elegant method termed specific hybridization internalization probe (SHIP) assay to differentiate between internalised and non-internalised material in live cells[15]. This assay utilises a short (20-mer) single-stranded DNA (ssDNA) fluorescent internalization probe (FIP) coupled to the ligand of interest and a complementary ssDNA quenching probe (QP). The SHIP system has been used almost exclusively in high-throughput flow cytometry studies, leaving its potential for imaging unexplored
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