Abstract
Measuring changes in intracellular Ca2+ concentration ([Ca2+] i ) with optical methods is a useful approach to study the function and regulation of Ca2+-permeable ion channels, such as ionotropic glutamate receptors. Here we describe a practical method for monitoring changes in [Ca2+] i that employs the widely used Ca2+ indicator, fura-2. Upon binding Ca2+, the excitation maximum of fura-2 shifts from 380 to 340 nm. Therefore, the ratio of fura-2 fluorescence from cells excited at 340 relative to 380 nm tracks changes in [Ca2+] i ; importantly this ratio is independent of dye concentration, optical path length, or illumination intensity. We provide instructions for calibrating an imaging system and using ratio-metric analysis for processing fura-2 fluorescence images to represent [Ca2+] i . Common technical problems are discussed in a section devoted to troubleshooting. Fura-2-based digital imaging has become a widely used technique with broad applicability. We describe methods to accomplish particularly common [Ca2+] i imaging goals; however, these provide a versatile foundation that can be further developed into more complex approaches to acquire [Ca2+] i -dependent images with higher temporal or spatial resolution, and from more challenging preparations.
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Acknowledgement
This work was supported by NIH grant DA07304 from the National Institute on Drug Abuse to SAT. Dr. Krogh was supported by NIDA training grant DA007097.
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Krogh, K.A., Thayer, S.A. (2016). Calcium Imaging to Study NMDA Receptor-mediated Cellular Responses. In: Popescu, G. (eds) Ionotropic Glutamate Receptor Technologies. Neuromethods, vol 106. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2812-5_15
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DOI: https://doi.org/10.1007/978-1-4939-2812-5_15
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2811-8
Online ISBN: 978-1-4939-2812-5
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