QUANTITATIVE INDO-1 MICROBEAD STANDARD IN FLOW CYTOMETRY

细胞内钙离子浓度是已知的发挥重要作用 在生理和病理生理条件下。 最近，许多 已经注意到测定细胞内游离的 使用流式细胞术测定钙浓度。 新开发的荧光 探针，如表现出大的光谱发射位移的Indo-1 当它螯合Ca++离子时， 这个应用程序。 然而，由于流式细胞仪的校准 相对于FL 1（405 nm峰）和FL 2比值的响应 (485 nm峰）细胞计数术受到限制。 本提案的重点 开发Indo-1微珠标准品试剂盒， Indo-1包被的微珠悬浮在一系列溶液中，所述溶液含有 预定浓度的游离Ca++。 使用该试剂盒， Indo-1微珠FL 1和FL 2发射荧光可以 与游离钙浓度直接相关。 这样的套件不会 不仅使流式细胞仪的校准更方便，而且还 有助于规范数据随时间的变化和实验室之间的数据。 更准确的 细胞溶质钙浓度的测定无疑将改善 我们对基因改变的生物学意义的理解 细胞内钙浓度

Intracellular calcium concentration is known to play a significant role in both physiological and pathophysiological conditions. Recently, much attention has been given to the determination of free intracellular calcium concentration using flow cytometry. Newly developed fluorescent probes, such as Indo-1 which exhibits a large spectral emission shift when it chelates Ca++ ions have been instrumental in the development of this application. However, because calibration of the flow cytometer response with respect to the ratio of the FL1 (405 nm peak) and the FL2 (485 nm peak) cytometry has been limited. The present proposal focuses on the development of an Indo-1 Microbead Standards Kit consisting of Indo-1 coated microbeads suspended in a series of solutions containing pre-determined concentrations of free Ca++. Using this kit the ratio of the FL1 and FL2 emission fluorescence of Indo-1 microbeads can be directly correlated with free calcium concentration. Such a kit will not only make calibration of flow cytometers more convenient, but will also help to normalize data over time and among laboratories. A more accurate determination of cytosolic calcium concentration will undoubtedly improve our understanding of the biological relevance of alterations in intracellular calcium concentration.