Verifying the Function and Localization of Genetically Encoded Ca2+Sensors and Converting FRET Ratios to Ca2+Concentrations

J. Genevieve Park, Amy E. Palmer
2015 Cold Spring Harbor Protocols  
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
more » ... Ca 2+ concentrations. Materials Reagents ATP stock solution for ER sensor calibrations Calcium chloride stock solution (1 M) in water Chelex® 100 resin, sodium form, 50-100 mesh Chelex®-treated double deionized H 2 O All solutions in this protocol should be prepared using Chelex®treated water and should be stored in metal-free plastic containers. Digitonin stock solution EGTA stock solution HEPES-buffered Hanks balanced salt solution (HHBSS) with Ca 2+ HEPES-buffered Hanks balanced salt solution without Ca 2+ (Ca 2+ -free HHBSS) Ionomycin stock solution . Mammalian expression plasmid encoding the ratiometric, genetically-encoded Ca 2+ biosensor of choice
doi:10.1101/pdb.prot076547 pmid:25561614 pmcid:PMC4968932 fatcat:z66d7fvpqjecnlhrtrak73zna4