A comparison of fluorescent Ca²⁺ indicators for imaging local Ca²⁺ signals in cultured cells.
A comparison of fluorescent Ca²⁺ indicators for imaging local Ca²⁺ signals in cultured cells.
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DOI：
10.1016/j.ceca.2015.10.003
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发表时间：
2015-12
期刊：
影响因子：
4
通讯作者：
Smith IF
中科院分区：
文献类型：
--
作者：
Lock JT;Parker I;Smith IF
Ca 2+的局部亚细胞变化是重要的细胞信号传导元件，调节神经元兴奋性和基因表达等多种过程。荧光Ca 2+指示剂的可用性极大地促进了细胞Ca 2+信号传导的研究。不同的指标来监测细胞内Ca 2+水平的整体变化的各自优点已被广泛评估，但它们在检测和分析局部，亚细胞Ca 2+信号的使用缺乏全面的比较。在这里，我们使用高速视频显微镜，在培养的人神经母细胞瘤SH-SY 5 Y细胞中，在1，4，5-三磷酸肌醇（IP 3）诱发的局部Ca 2+信号（脉冲）的背景下评估了几种荧光Ca 2+指示剂。总共，九种合成的Ca 2+染料（Fluo-4、Fluo-8、Fluo-8高亲和力、Fluo-8低亲和力、俄勒冈州绿色BAPTA-1、Cal-520、Rhod-4、Asante钙红和X-Rhod-1）和三种遗传编码的Ca 2+指示剂（GCaMP 6-慢、-中和-快变体）进行了测试;标准包括局部Ca 2+喷流的幅度、动力学、信噪比和检测效率。其中，我们得出的结论是，Cal-520是检测和忠实跟踪局部事件的最佳指示剂; Rhod-4是首选的红色发光指示剂;并且没有一种GCaMP 6变体非常适合亚细胞Ca 2+信号成像。
Localized subcellular changes in Ca2+ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca2+ signaling have been greatly facilitated by the availability of fluorescent Ca2+ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca2+ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca2+ signals is lacking. Here, we evaluated several fluorescent Ca2+ indicators in the context of local Ca2+ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP3) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca2+ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca2+-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca2+ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca2+ signals.