Ca2+-Imaging Techniques to Analyze Ca2+ Signaling in Cells and to Monitor Neuronal Activity in the Retina
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Ca2+ is an important regulator of many cell functions including proliferation, apoptosis, movements, secretion, contraction, excitation, and differentiation. The regulation of these different cell functions is encoded by the specific temporal and spatial distribution of Ca2+ signals. In degenerative diseases mutations can lead to changes in cell functions in the worst case to apoptosis. Thus analysis of signals arising as changes in intracellular free Ca2+ represent an important step towards the understanding of mutation-dependent or environmental impact into cell function. The classic approach to study changes in intracellular free Ca2+ is the measurement of intracellular Ca2+ by using Ca2+ -sensitive fluorescence dyes in conjunction with fluorescence microscopy as a method called Ca2+ imaging.
In this chapter the basic method and a short theoretical background will be provided to perform Ca2+ -imaging experiments. As a model cultured retinal pigment epithelial cells will be used. The basic steps of the method are the loading of the cells with the fluorescence dye by incubation with a membrane permeable ester of the dye. The next step would be the application of an agonist which can be further analyzed by blockers of enzymes or by manipulating the different Ca2+ -storing compartments which contribute to changes in intracellular free Ca2+ . At the end of an experiment an on-cell type of calibration will be performed to calculate the underlying concentration of intracellular free Ca2+ . Furthermore, the successful calibration of an experiment can be used as a measure of a reliable experiment. In addition to that, three examples for basic experiments will be given which can lead to a first insight into the mechanism underlying changes in cytosolic free Ca2+ as a second messenger.
