USE OF TIME-RESOLVED FLUORESCENCE TO DETERMINE INTRACELLULAR ION GRADIENTS

荧光探针Quin-2已被广泛用于检测 稳态荧光法测定细胞内钙离子浓度 测量。利用纳秒时间分辨脉冲和相位荧光法 我们已经确定这个探测器表现出独特的衰变 结合各种阳离子的特性，包括Ca++，Mg++和 锌离子；具体地说，它显示出强度增加了五到十倍， 以及各向异性的变化。研究相互作用的测量方法 在溶液中Quin-2与阳离子络合物之间的相互作用表明 自由探针和几种不同离子络合物的浓度可以是 解决了。我们已经获得了Quin-2的光谱和时间分辨数据- 加载A431细胞以检查细胞内的区隔作用 细胞因子结合诱导的信号转导事件的功能。从… 我们的数据支持Quin-2存在于非均质 细胞微环境我们正在开发一种依赖生长因子的 细胞的细胞质功能图谱。

The fluorescent probe Quin-2 has been used extensively to determine intracellular calcium ion concentrations by steady-state fluorescence measurements. Using nanosecond time-resolved pulse and phase fluorometric instrumentation we have determined that this probe exhibits unique decay characteristics upon binding various cations including Ca++, Mg++, and Zn++; specifically, it shows a five- to ten-fold increase in intensity as well as alterations in anisotropy. Measurements to study the interactions between Quin-2 and cation complexes in solution demonstrate that concentrations for free probe and several different ion complexes can be resolved. We have obtained spectral and time-resolved data from Quin-2- loaded A431 cells to examine intracellular compartmentalization as a function of signal transductory events induced by cytokine binding. From our data which support the idea that Quin-2 exists in heterogeneous cellular microenvironments we are developing a growth factor-dependent cytoplasmic functional map of the cell.