Two dye two wavelength excitation calcium imaging: results from bovine adrenal chromaffin cells.
Cell Calcium
; 24(1): 71-84, 1998 Jul.
Article
en En
| MEDLINE
| ID: mdl-9793690
ABSTRACT
We tested a mixture of Calcium-Green-1 (CG-1) and Brilliantsulfaflavine (BS) for dual excitation ratiometric measurements of the intracellular free calcium concentration ([Ca2+]i) in bovine adrenal chromaffin cells. Dyes were coloaded (without being molecularly linked to each other) in the whole-cell configuration of the patch clamp technique. We compared the loading time-courses of CG-1 and BS, investigated their intracellular distribution patterns and studied the time course of photobleaching. We determined the apparent dissociation constant of CG-1, both optically and by potentiometric titration. Our findings indicate that (i) with excitation at 420/488 nm, calibrated fluorescence signals could be derived using a Grynkiewicz-type equation; (ii) BS is an ideal reference dye that displayed no interaction with CG-1 or cellular constituents; and (iii) that calibration requires diffusional equilibration between pipette and the accessible volume of the cell. Spatially resolved recordings of fluorescence excitation spectra revealed elevated fluorescence of CG-1 in the nucleus such that reported [Ca2+]i levels seemed 25% higher compared to cytosolic values. Comparing fluorescence emission from in vitro dye solutions with in vivo values, we could estimate the accessible volume fraction and amount of Ca(2+)-insensitive dye.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Calcio
/
Glándulas Suprarrenales
/
Células Cromafines
/
Colorantes Fluorescentes
/
Isoquinolinas
Límite:
Animals
Idioma:
En
Revista:
Cell Calcium
Año:
1998
Tipo del documento:
Article
País de afiliación:
Alemania