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Verifying the function and localization of genetically encoded Ca2+ sensors and converting FRET ratios to Ca2+
J Genevieve Park1, Amy E Palmer1
1Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309.
Abstract:
Genetically encoded, ratiometric, fluorescent Ca(2+) biosensors can be used in living cells to quantitatively measure free Ca(2+) concentrations in the cytosol or in organelles. This protocol describes how to perform a calibration of a Ca(2+) sensor expressed in cultured mammalian cells as images are acquired using a widefield fluorescence microscope. This protocol also explains how to calculate Förster resonance energy transfer (FRET) ratios from acquired images and how to convert FRET ratios to Ca(2+) concentrations.
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