USE OF TIME-RESOLVED FLUORESCENCE TO DETERMINE INTRACELLULAR ION GRADIENTS

使用时间分辨荧光测定细胞内离子梯度

• 批准号: 3770353
• 负责人: B PACKARD
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3770353
• 关键词: biological signal transduction; calcium indicator; cations; cytology; cytoplasm; fluorescence spectrometry; fluorescent dye /probe; growth factor; intracellular; ionic strengths; time resolved data

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.

荧光探针Quin-2已被广泛用于测定 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 我们已经确定这个探测器表现出独特的衰变 在结合各种阳离子（包括Ca++、Mg++和 Zn++;具体地，它显示出强度增加五到十倍， 以及各向异性的改变。 研究相互作用的测量 Quin-2和溶液中阳离子络合物之间的关系表明， 游离探针和几种不同离子复合物的浓度可以 解决了 我们已经获得了光谱和时间分辨数据从奎因-2- 加载A431细胞，以检查细胞内区室化， 由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存在于异构体中的想法， 我们正在开发一种生长因子依赖的细胞微环境， 细胞的细胞质功能图。