Advances in the use of the fluorescent probe fura-2 for the estimation of intrasynaptosomal calcium.
Fura-2 has been used to measure intracellular Ca2+ 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 Ca2+ ([Ca2+]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 [Ca2+]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 [Ca2+]i without compromising membrane integrity.
Yates
S L
SL
Duke University Medical Center, Integrated Toxicology Program, Durham, North Carolina.
Fluhler
E N
EN
Lippiello
P M
PM
D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002540:Cerebral Cortex / Q000276:immunology / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005260:Female; D016257:Fura-2; D066298:In Vitro Techniques; D008823:Micelles; D011060:Poloxalene / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012710:Serum Albumin, Bovine / Q000276:immunology / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013574:Synaptosomes / Q000737:chemistry* / Q000378:metabolism
D003902:Detergents; D008823:Micelles; D012710:Serum Albumin, Bovine; D011189:Potassium Chloride; D011060:Poloxalene; D002118:Calcium; D016257:Fura-2