B2 Book chapter

Analysis of intracellular vesicles in B lymphocytes: antigen traffic in the spotlight

List of Authors: Hernández-Pérez Sara, Runsala Marika, Šuštar Vid, Mattila Pieta K.

Publisher: Humana Press Inc.

Publication year: 2021

Book title *: Confocal Microscopy: Methods and Protocols

Journal name in source: Methods in Molecular Biology

Title of series: Methods in Molecular Biology

Volume number: 2304

ISBN: 978-1-0716-1401-3

eISBN: 978-1-0716-1402-0

ISSN: 1064-3745

DOI: http://dx.doi.org/10.1007/978-1-0716-1402-0_9


All eukaryotic cells are delimited by the plasma membrane, separating the cell from its environment. Two critical cellular pathways, the endocytic and the exocytic vesicle networks, shuttle material in and out the cell, respectively. The substantial development of cell biological imaging techniques, along with improved fluorescent probes and image analysis tools, has been instrumental in increasing our understanding of various functions and regulatory mechanisms of various intracellular vesicle subpopulations and their dynamics. Here, using B lymphocytes (B cells) as a model system, we provide a protocol for 3D analysis of the intracellular vesicle traffic in either fixed or living cells using spinning disk confocal microscopy. We also describe the usage of image deconvolution to improve the resolution, particularly important for vesicular networks in lymphocytes due to the small size of these cells. Lastly, we describe two types of quantitative analysis: vesicle distribution/clustering toward the microtubule organizing center (MTOC), and colocalization analysis with endolysosomal markers.

Last updated on 2021-24-06 at 09:09