Analysis of Intracellular Vesicles in B Lymphocytes: Antigen Traffic in the Spotlight

Hernández-Pérez, Sara; Runsala, Marika; Šuštar, Vid; Mattila, Pieta K.

doi:10.1007/978-1-0716-1402-0_9

Cited by 1 publication

(1 citation statement)

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“…Cells were then washed with PBS to remove excess unbound antigen and resuspended in Imaging Buffer (10 6 /ml). SDCM and Airyscan: After washing, cells (2 × 10 4 /well) were activated for different time points in an incubator (5% CO 2 , 37ºC) in a 12-well fibronectin-coated (80 ng/ well) PTFE diagnostic slide (Fisher Scientific, 10028210) 22 . After activation, internalisation was stopped by placing the samples on ice.…”

Section: Microscopy Samplesmentioning

confidence: 99%

A specific hybridisation internalisation probe (SHIP) enables precise live-cell and super-resolution imaging of internalized cargo

Hernández-Pérez

Mattila

2022

Sci Rep

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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.

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Section: Microscopy Samplesmentioning

confidence: 99%