A reevaluation of neuronal zinc measurements: Artifacts associated with high intracellular dye concentration
A reevaluation of neuronal zinc measurements: Artifacts associated with high intracellular dye concentration
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DOI：
10.1124/mol.62.3.618
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发表时间：
2002-09-01
影响因子：
3.6
通讯作者：
Reynolds, IJ
中科院分区：
文献类型：
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作者：
Dineley, KE;Malaiyandi, LM;Reynolds, IJ
锌作为一种强有力的神经毒素的出现促进了适用于测量培养细胞中细胞内游离锌（[Zn 2 +]（i））的技术的发展。因此，一个新的家族的Zn 2+敏感的荧光团已经变得可用。利用培养神经元中离子载体诱导的[Zn 2 +]（i）升高，我们测量了新型染料FuraZin-1和FluoZin-2中[Zn 2 +]（i）诱导的变化，并将其与已建立的[Zn 2 +]（i）敏感荧光团mag-fura-2和纽波特绿色进行比较。所有这些染料都能有效地检测[Zn 2 +]（i），并且FuraZin-1、FluoZin-2和纽波特绿色对[Zn 2 +]（i）的选择性优于[Ca 2 +]（i）和[Mg 2 +]（i）。然而，染料在它们对[Zn 2 +]（i）的表观灵敏度上显示出很小的差异，即使它们对Zn 2+的体外亲和力从20 nM变化到3 μ M。我们在此表明，这是在这种性质的实验中使用的相对高浓度的细胞内染料的结果。因此，对于[Zn 2 +]（i）的测量，报告系统的灵敏度由细胞内染料浓度主导，而染料亲和力不重要。我们扩展这些研究结果表明，校准的染料信号离子浓度是非常依赖于细胞内染料浓度的精确测量。
The emergence of zinc as a potent neurotoxin has prompted the development of techniques suitable for the measurement of intracellular free zinc ([Zn2+](i)) in cultured cells. Accordingly, a new family of Zn2+-sensitive fluorophores has become available. Using ionophore-induced elevations of [Zn2+](i) in cultured neurons, we measured [Zn2+](i)-induced changes in the novel dyes FuraZin-1 and FluoZin-2 and compared them with the established [Zn2+](i)-sensitive fluorophores mag-fura-2 and Newport Green. All of these dyes effectively detected [Zn2+](i), and FuraZin-1, FluoZin-2, and Newport Green showed selectivity for [Zn2+](i) over [Ca2+](i) and [Mg2+](i). However, the dyes showed little difference in their apparent sensitivity to [Zn2+](i), even though their in vitro affinities for Zn2+ varied from 20 nM to 3 muM. We show herein that this is a consequence of the relatively high concentrations of intracellular dye used in experiments of this nature. Thus, for the measurement of [Zn2+](i), the sensitivity of the reporting system is dominated by the intracellular dye concentration, whereas dye affinity is unimportant. We extend these findings to show that calibration of dye signal to ion concentration is critically dependent on precise measurement of intracellular dye concentration.