title: A comparison of fluorescent Ca²⁺ indicators for imaging local Ca²⁺ signals in cultured cells.
abstract: Localized subcellular changes in Ca(2+) serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca(2+) signaling have been greatly facilitated by the availability of fluorescent Ca(2+) indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca(2+) levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca(2+) signals is lacking. Here, we evaluated several fluorescent Ca(2+) indicators in the context of local Ca(2+) signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP3) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca(2+) dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca(2+)-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca(2+) puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca(2+) signals.
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine, CA, United States. Electronic address: lockj@uci.edu.
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Lock
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine, CA, United States; Department of Physiology and Biophysics, University of California, Irvine, CA, United States. Electronic address: iparker@uci.edu.
authors.forename: Ian
authors.initials: I
authors.lastname: Parker
authors.affiliation: Department of Neurobiology and Behavior, University of California, Irvine, CA, United States. Electronic address: ismith@uci.edu.
authors.forename: Ian F
authors.initials: IF
authors.lastname: Smith
mesh_terms: D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D007700:Kinetics
chemical_list: D005456:Fluorescent Dyes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium
keywords: Calcium; Fluorescent indicators; GCaMP6; Genetically encoded calcium indicators; Local calcium signals