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High-Resolution Imaging of Intracellular Trafficking of B Cell Receptor Using Specific Hybridization Internalization Probe (SHIP)
Tekijät: Hernández-Pérez, Sara, Mattila, Pieta K.
Toimittaja: Palash Chandra Maity
Kustantaja: Humana Press Inc.
Julkaisuvuosi: 2025
Kokoomateoksen nimi: B-Cell Receptor Signaling - Methods and Protocols
Tietokannassa oleva lehden nimi: Methods in Molecular Biology
Sarjan nimi: Methods in Molecular Biology
Vuosikerta: 2909
Aloitussivu: 73
Lopetussivu: 82
ISBN: 978-1-0716-4441-6
eISBN: 978-1-0716-4442-3
ISSN: 1064-3745
eISSN: 1940-6029
DOI: https://doi.org/10.1007/978-1-0716-4442-3_6
Verkko-osoite: https://link.springer.com/protocol/10.1007/978-1-0716-4442-3_6
Tiivistelmä
Recent advancements in microscopy have greatly expanded our understanding of intracellular traffic. Yet, due to the inherent characteristics of B cells, such as their small size and high receptor density on the plasma membrane, visualization of internalized cargo or receptors remains challenging. This challenge is particularly pronounced in the case of the B cell receptor (BCR), where accurate detection of internalized, antigen-bound BCR molecules can be strongly hindered by the signal from the plasma membrane-bound pool of the same molecules. To tackle this issue, we adapted the Specific Hybridization Internalization Probe (SHIP) assay, initially designed for flow cytometry studies, for the study of BCR internalization using microscopy. This assay utilizes a single-stranded DNA (ssDNA) fluorescence internalization probe (FIP) paired with a complementary ssDNA quenching probe that “turns off” the signal from the (extracellular) surface-bound BCRs, greatly facilitating the unambiguous identification of internalized (intracellular) receptors. Moreover, the assay is versatile and adaptable to a range of imaging modalities, including live-cell imaging and super-resolution microscopy. SHIP proves to be a valuable tool in the study of intracellular processes, offering enhanced imaging precision for the detection of internalized BCRs.
Recent advancements in microscopy have greatly expanded our understanding of intracellular traffic. Yet, due to the inherent characteristics of B cells, such as their small size and high receptor density on the plasma membrane, visualization of internalized cargo or receptors remains challenging. This challenge is particularly pronounced in the case of the B cell receptor (BCR), where accurate detection of internalized, antigen-bound BCR molecules can be strongly hindered by the signal from the plasma membrane-bound pool of the same molecules. To tackle this issue, we adapted the Specific Hybridization Internalization Probe (SHIP) assay, initially designed for flow cytometry studies, for the study of BCR internalization using microscopy. This assay utilizes a single-stranded DNA (ssDNA) fluorescence internalization probe (FIP) paired with a complementary ssDNA quenching probe that “turns off” the signal from the (extracellular) surface-bound BCRs, greatly facilitating the unambiguous identification of internalized (intracellular) receptors. Moreover, the assay is versatile and adaptable to a range of imaging modalities, including live-cell imaging and super-resolution microscopy. SHIP proves to be a valuable tool in the study of intracellular processes, offering enhanced imaging precision for the detection of internalized BCRs.
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