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Advances in the use of the fluorescent probe fura‐2 for the estimation of intrasynatposomal calcium
Author(s) -
Yates S. L.,
Fluhler E. N.,
Lippiello P. M.
Publication year - 1992
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.490320215
Subject(s) - fura 2 , micelle , membrane , chemistry , bovine serum albumin , lipophilicity , biophysics , intracellular , fluorescence , calcium , cytoplasm , biochemistry , chromatography , aqueous solution , organic chemistry , biology , enzyme , physics , quantum mechanics , cytosol
Fura‐2 has been used to measure intracellular Ca 2+ with great success in a variety of cell and subcellular preparations, including synaptosomes. There is however, a great deal of variability in the reported estimates of resting intrasynaptosomal Ca 2+ ([Ca 2+ ] i ). Fura‐2 AM is highly lipophilic and passes readily across the plasma membrane into the cytoplasm, where it is de‐esterified and trapped. The lipophilicity of fura‐2, however, promotes the formation of micelles in aqueous media, which may impede the passage of the probe across cell membranes. Our results suggest that some of the variability in the reported [Ca 2+ ] i estimates may be related to fura‐2 de‐esterification and loading efficiencies. The use of the nonionic detergent pluronic F‐127 is recommended to prevent the formation of fura‐2 micelles. The use of a detergent is not always an acceptable practice, however, especially in studies in which detergent–lipid interactions may influence membrane parameters. We found that fatty acid free bovine serum albumin (BSA) (0.25%) greatly increases the intrasynaptosomal concentration of the probe, resulting in a significant increase in the signal‐to‐noise (S/N) ratio. The mechanism appears to be independent of effects of BSA on synaptosomal integrity and directly related to the prevention of fura‐2 micelle formation, as evidenced by light spectroscopic scattering measurements. Thus, BSA appears to keep the probe in a form that crosses the synaptic plasma membrane more readily. The effectiveness of BSA in improving the loading of fura‐2 into synaptosomes was comparable to the detergent pluronic F‐127, making it possible to measure [Ca 2+ ] i without compromising membrane integrity. © 1992 Wiley‐Liss, Inc.