title: Verifying the function and localization of genetically encoded Ca2+ sensors and converting FRET ratios to Ca2+ concentrations.
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.
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309.
authors.forename: J Genevieve
authors.initials: JG
authors.lastname: Park
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Palmer
mesh_terms: D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells / Q000166:cytology; D006801:Humans; D052139:Intracellular Calcium-Sensing Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D015388:Organelles / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D014162:Transfection
chemical_list: D052139:Intracellular Calcium-Sensing Proteins; D002118:Calcium