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Visualizing formation and dynamics of vacuoles in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles

Voronina, S. G., Sherwood, M. W., Gerasimenko, Oleg Vsevolodovich, Petersen, Ole Hoger and Tepikin, A. V. 2007. Visualizing formation and dynamics of vacuoles in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles. American Journal of Physiology-Gastrointestinal and Liver Physiology 293 (6) , G1333-G1338. 10.1152/ajpgi.00275.2007

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Abstract

Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualized by confocal microscopy. A combination of two dextran-bound probes, one infused into the cytosol and the second added to the extracellular solution, was used to identify endocytic and nonendocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localization of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosolic dextran-bound probes with endoplasmic reticulum (ER) or mitochondrial probes were also used to simultaneously visualize vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Systems Immunity Research Institute (SIURI)
Publisher: American Society for Biochemistry and Molecular Biology
ISSN: 0193-1857
Last Modified: 04 Jun 2017 06:31
URI: http://orca-mwe.cf.ac.uk/id/eprint/61322

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