Yates S L, Fluhler E N, Lippiello P M
Duke University Medical Center, Integrated Toxicology Program, Durham, North Carolina.
J Neurosci Res. 1992 Jun;32(2):255-60. doi: 10.1002/jnr.490320215.
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.
在包括突触体在内的各种细胞和亚细胞制剂中，Fura-2已被成功用于测量细胞内Ca2+。然而，据报道，静息突触体内Ca2+（[Ca2+]i）的估计值存在很大差异。Fura-2 AM具有高度亲脂性，很容易穿过质膜进入细胞质，在那里它被去酯化并被困住。然而，fura-2的亲脂性会促进其在水性介质中形成胶束，这可能会阻碍探针穿过细胞膜。我们的结果表明，报道的[Ca2+]i估计值的一些差异可能与fura-2的去酯化和加载效率有关。建议使用非离子洗涤剂普朗尼克F-127来防止fura-2胶束的形成。然而，使用洗涤剂并不总是一种可接受的做法，特别是在洗涤剂-脂质相互作用可能影响膜参数的研究中。我们发现，不含脂肪酸的牛血清白蛋白（BSA）（0.25%）可大大提高突触体内探针的浓度，从而导致信噪比（S/N）显著增加。光散射光谱测量表明，其机制似乎与BSA对突触体完整性的影响无关，而直接与防止fura-2胶束形成有关。因此，BSA似乎使探针保持在更容易穿过突触质膜的形式。BSA在提高fura-2加载到突触体中的有效性与洗涤剂普朗尼克F-127相当，从而可以在不损害膜完整性的情况下测量[Ca2+]i。
