NDLI: Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry.

Content Provider	PubMed Central
Author	Brandes, R. Camacho, S. A. Baker, A. J. Weiner, M. W. Figueredo, V. M.
Abstract	Fluorometric determination of cytosolic calcium, [Ca2+]c, using Indo-1 in intact tissue, is limited by problems in obtaining calibration parameters for Indo-1 in vivo. Therefore, the goal of this study was to calibrate Indo-1 using in vitro constants, obtained from protein-containing reference solutions designed to produce similar Indo-1 spectral properties to those in vivo. Due to wavelength-dependent tissue light absorbance, the in vitro constants had to be absorbance-corrected using a novel method. The correction factor was calculated from the relationship between the Indo-1 fluorescence intensities at the two detection wavelengths. A mixture of proteins at approximately 28 mg/ml had a similar Indo-1 isosbestic wavelength (430 nm) to that found in vivo (427 nm), and a similar fluorescence ratio maximum with saturating Ca2+ to that found in vivo (after absorbance correction). Using calibration constants from this protein mixture, calculated [Ca2+]c in a Langendorf perfused rat heart was 187 nM during diastole, and 464 nM in systole. This new calibration method circumvented the considerable experimental problems of previous methods which required measurements with the cytosol fully depleted and fully saturated with Ca2+.
Related Links	http://dx.doi.org/10.1016/s0006-3495(93)81274-8
Ending Page	1982
Page Count	10
Starting Page	1973
File Format	PDF
ISSN	00063495
e-ISSN	15420086
Journal	Biophysical Journal
Issue Number	5
Volume Number	65
Language	English
Publisher Date	1993-11-01
Access Restriction	Open
Subject Keyword	Biophysics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Biophysics

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