Measuring calcium signaling using genetically targetable fluorescent indicators
Measuring calcium signaling using genetically targetable fluorescent indicators
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DOI：
10.1038/nprot.2006.172
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Tsien, Roger Y.
中科院分区：
文献类型：
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作者：
Palmer, Amy E.;Tsien, Roger Y.
遗传编码的钙(2+)指示剂允许研究人员在各种实验系统中定量测量钙(2+)的动态。该方案总结了可用的指示剂，并强调了最适合一些实验条件的指示剂，例如测量特定细胞器中的Ca(2+)和哺乳动物组织培养细胞中的定位。该方案本身侧重于骆驼的使用，它是一种基于荧光共振能量转移(FRET)的指示剂，由两种荧光蛋白和两种钙反应元件(钙调素(CaM)的变体和CaM结合肽)组成。该方案详细介绍了如何建立和进行Ca(2+)成像实验，完成离线数据处理(如背景校正)，并将观测到的FRET比率变化转换为Ca(2+)浓度。此外，我们强调了在观察细胞器钙(2+)方面的一些挑战，以及研究人员可以采用的替代策略，以有效地校准这些位置的遗传编码的钙(2+)指示物。假设所有部件都随时可用，设置和进行显微镜系统的初始校准估计需要大约1周时间。细胞培养和转染估计需要类似于3d的时间(从将细胞接种到成像盘上的时间)。一次实验和校准可能需要几个小时。最后，根据分析程度的不同，离线数据处理可能需要大约1天的时间。
Genetically encoded Ca(2+) indicators allow researchers to quantitatively measure Ca(2+) dynamics in a variety of experimental systems. This protocol summarizes the indicators that are available, and highlights those that are most appropriate for a number of experimental conditions, such as measuring Ca(2+) in specific organelles and localizations in mammalian tissue-culture cells. The protocol itself focuses on the use of a cameleon, which is a fluorescence resonance-energy transfer (FRET)-based indicator comprising two fluorescent proteins and two Ca(2+)-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). This protocol details how to set up and conduct a Ca(2+)-imaging experiment, accomplish offline data processing (such as background correction) and convert the observed FRET ratio changes to Ca(2+) concentrations. Additionally, we highlight some of the challenges in observing organellar Ca(2+) and the alternative strategies researchers can employ for effectively calibrating the genetically encoded Ca(2+) indicators in these locations. Setting up and conducting an initial calibration of the microscope system is estimated to take similar to 1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take similar to 3 d (from the time of plating cells on imaging dishes). An experiment and calibration will probably take a few hours. Finally, the offline data workup can take similar to 1 d depending on the extent of analysis.