A new technique for assessing the microscopic distribution of cellular calcium exit sites

This paper contains a description of a new method designed to monitor the distribution of Ca2+ efflux from cells or small cellular aggregates. The idea behind this method is to use a fluorescent Ca2+ indicator bound to dextrans of high molecular weight to slow down Ca2+ diffusion. Due to the decrease in diffusion rate, Ca2+ ions should be held close to the site of their release from the cells for a relatively long time, enough for the confocal microscope to detect such a local increase in Ca2+ concentration. This paper gives a detailed description of the method, illustrated with results of measurements of agonist-dependent and agonist-independent Ca2+ extrusion from pancreatic acinar cells. An appendix provides the mathematical background that should allow selection of the concentration of buffer which is necessary to achieve a particular Ca2+ diffusion coefficient.