Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity Calcium Ruby and its dextran conjugate

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TitreMeasuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity Calcium Ruby and its dextran conjugate
Type de publicationJournal Article
Nouvelles publications2009
AuteursLuccardini, C, Yakovlev AV, Pasche M, Gaillard S, Li DD, Rousseau F, Ly R, Becherer U, Mallet J-M, Feltz A, Oheim M
JournalCell Calcium
Volume45
Fascicule3
Pagination275-283
Année de publicationMar
Numéro0143-4160
Accession NumberISI:000264956800009
Résumé

The limited choice and poor performance of red-emitting calcium (Ca2+) indicators have hampered microfluorometric measurements of the intracellular free Ca2+ concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca2+ indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH2 linker arm. The low-affinity variant (K-D.Ca, similar to 30 mu M) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca2+ transients. While Calcium Ruby is a mitochondrial Ca2+ probe, its conjugation, via the NH2 tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a Cytosolic Call probe with an affinity matched to microdomain Ca2+ signals. As an example, we show depolarization-evoked Ca2+ signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca2+ measurements. (c) 2008 Elsevier Ltd. All rights reserved.

URL<Go to ISI>://000264956800009
DOI10.1016/j.ceca.2008.11.007
Importer un fichierMeasuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity Calcium Ruby and its dextran conjugate
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