Measuring mitochondrial and cytoplasmic Ca2+ in EGFP expressing cells with a low-affinity calcium Ruby and its dextran conjugate.
Cell Calcium
; 45(3): 275-83, 2009 Mar.
Article
en En
| MEDLINE
| ID: mdl-19167753
The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) 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 NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) 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 Ca(2+) measurements.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Rodaminas
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Calcio
/
Dextranos
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Células Cromafines
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Citoplasma
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Proteínas Fluorescentes Verdes
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Mitocondrias
Límite:
Animals
Idioma:
En
Revista:
Cell Calcium
Año:
2009
Tipo del documento:
Article
País de afiliación:
Francia
Pais de publicación:
Países Bajos