MEASURE OF PLATELET INTRACELLULAR FREE CALCIUM ION CONCENTRATION: CELL BEHAVIOR

比率荧光显微镜被用来成像和测量 细胞内游离钙离子浓度，[Ca++]i 在生物材料基质上附着和扩散的血小板。这个 通过对基质的组成和化学成分进行了表征。 艾斯卡。负载了一种新型的钙离子敏感荧光团--富拉红(Fura Red，tm) 进入血小板，并实现了系统的现场校准。 静息细胞内[Ca++]i为50~100 nm。[Ca++]i 对物质接触的反应被发现是不同的。跟随 在长达5分钟的可变滞后时间内，响应细胞表现出 [Ca++]i升高100-300 nm以上。其他血小板显示很少或 与表面接触时，[Ca++]i无明显变化。那些血小板 显示峰值[Ca++]i大于350 Nm，发现 在实验期间传播了，而那些没有传播的 [Ca++]i值保持在250 Nm以下； 血小板扩散与[Ca++]i升高有关。 还对附着在表面的血小板进行了成像。 流动细胞悬浮液。研究发现，血小板附着在 显示出伪足形成的早期迹象，没有明显的 在开始的几分钟内升高[Ca++]i 联系。结果显示了一种新的可视化方法 接触刺激下血小板的信号转导过程 为进一步研究[Ca++]i奠定了基础 血小板内的信号转导与[Ca++]i的可能相关性 对血小板活化的其他方面的反应，如颗粒 具有释放和促进凝血作用。

Ratio fluorescence microscopy has been used to image and measure the intracellular free calcium concentration, [Ca++]i, in single platelets as they attach and spread on biomaterial substrates. The composition and chemistry of the substrates were characterized by ESCA. A novel Ca++-sensitive fluorophore, Fura Red(tm) was loaded into platelets and an in situ calibration of the system was achieved. The [Ca++]i in resting cells was observed to be 50-100nM. The [Ca++]i response to material contact was found to be heterogeneous. Following a variable lag period of up to 5 minutes, responding cells exhibited a rise in [Ca++]i of 100-300nM more. Other platelets showed little or no change in [Ca++]i in contact with the surface. Those platelets demonstrating peak [Ca++]i values greater than 350nM were found to have spread during the experiment, while those that did not spread were seen to have [Ca++]i values remaining below 250nM; suggesting that platelet spreading is associated with a rise in [Ca++]i. Calcium imaging was also performed on platelets adhering to surfaces from a flowing cell suspension. It was found that platelets attached and showed early signs of pseudopodia formation without significant increases [Ca++]i during the initial few minutes of contact. The results demonstrate a novel method of visualizing the process of signal transduction in platelets stimulated by contact with biomaterials, and lay the groundwork for further study of [Ca++]i signaling in platelets and of the possible correlation of the [Ca++]i response with other aspects of platelet activation such as granule release and the promotion of blood coagulation.