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

in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles. Am J Physiol Gastrointest Liver Physiol 293: G1333–G1338, 2007. First published August 23, 2007; doi:10.1152/ajpgi.00275.2007.—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 concentra-tions of cholecystokinin (CCK). The vacuoles, forming in the cyto-plasm of the cell, were revealed as dark imprints on a bright fluores-cence 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, wa