Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3
Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3
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DOI：
10.1016/j.ceca.2004.10.003
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发表时间：
2005-03-01
期刊：
影响因子：
4
通讯作者：
Dineley, KE
中科院分区：
文献类型：
--
作者：
Devinney, MJ;Reynolds, IJ;Dineley, KE
细胞内游离锌（[Zn 2 +]（i））的升高可能有助于损伤范例中的细胞死亡，涉及钙失调和氧化应激，如谷氨酸兴奋性毒性。然而，很难在活细胞中同时监测这两种离子。在这里，我们提出了一种新的方法，使用荧光显微镜和离子敏感指示剂Fura-2FF和FluoZin-3来监测初级皮层神经元中的[Ca 2 +]（i）和[Zn 2 +]（i）。我们发现，新的单波长染料FluoZin-3对小锌负载的响应很强，对高Ca 2+或Mg 2+不敏感，并且相对不受低pH或氧化剂的影响。比率指示剂Fura-2FF对Ca ~（2+）和Zn ~（2+）均敏感。然而，在类似于兴奋性毒性谷氨酸暴露的条件下，其中[Ca 2 +]（i）相对于[Zn 2 +]（i）较高，我们发现fura-2FF主要对[Ca 2 +]（i）作出响应，但相对不受低[Zn 2 +]（i）的影响。此外，可以区分由高[Ca 2 +]（i）或高[Zn 2 +]（i）引起的fura-2FF比率变化，因为每种离子产生不同的光谱响应。最后，双染料实验表明，FluoZin-3和fura-2FF在强烈的谷氨酸暴露期间，在相同的神经元中分别对[Zn 2 +]（i）和[Ca 2 +]（i）产生强烈的响应。这些研究为同时检测细胞中的钙和锌提供了一种新的方法。(C)2004爱思唯尔有限公司保留所有权利。
Elevation of intracellular free zinc ([Zn2+](i)) probably contributes to cell death in injury paradigms involving calcium deregulation and oxidative stress such as glutamate excitotoxicity. However, it is difficult to monitor both ions simultaneously in live cells. Here we present a new method using fluorescence microscopy and the ion sensitive indicators fura-2FF and FluoZin-3 to monitor both [Ca2+](i) and [Zn2+](i) in primary cortical neurons. We show that the new single wavelength dye FluoZin-3 responds robustly to small zinc loads, is insensitive to high Ca2+ or Mg2+, and is relatively unaffected by low pH or oxidants. The ratiometric indicator fura-2FF is sensitive to both Ca2+ and Zn2+. However, in conditions analogous to excitotoxic glutamate exposure where [Ca2+](i) is high relative to [Zn2+](i) we found that fura-2FF responds mostly to [Ca2+](i) but is relatively unaffected by low [Zn2+](i). Moreover, fura-2FF ratio changes caused by high [Ca2+](i) or high [Zn2+](i) could be distinguished because each ion produces a different spectral response. Finally, dual dye experiments showed that FluoZin-3 and fura-2FF respond robustly to [Zn2+](i) and [Ca2+](i), respectively, in the same neurons during intense glutamate exposure. These studies provide a novel method for the simultaneous detection of both calcium and zinc in cells. (C) 2004 Elsevier Ltd. All rights reserved.