Tran Louie, Farinas Javier, Ruslim-Litrus Lily, Conley Pamela B, Muir Craig, Munnelly Kevin, Sedlock David M, Cherbavaz Diana B
Caliper Technologies, Mountain View, CA 94043, USA.
Anal Biochem. 2005 Jun 15;341(2):361-8. doi: 10.1016/j.ab.2005.02.024.
To facilitate drug discovery directed toward platelet-specific targets, we developed a platelet isolation and fluorophore-loading method that yields functionally responsive platelets in which we were able to detect agonist-induced calcium flux using a microfluidics-based screening platform. The platelet preparation protocol was designed to minimize preparation-induced platelet activation and to optimize signal strength. Measurement of platelet activation, as monitored by ratiometric determination of agonist-induced calcium flux in fluor-loaded human platelets, was optimized in a macrosample cuvette format in preparation for detection in a microfluidic chip-based assay. For the microfluidic device used in these studies, a cell density of 1 to 2 x 10(6) platelets per milliliter and a nominal flow rate of 5 to 10 nl per second provided optimal event resolution of 5 to 20 platelets traversing the detection volume per unit time. Platelets responded in a dose-dependent manner to adenosine diphosphate and protease-activating peptide (PAR) 1 thrombin receptor-activating peptide (TRAP). The work presented here constitutes proof-of-principle experiments demonstrating the enabling application of a microfluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets.
为了促进针对血小板特异性靶点的药物发现，我们开发了一种血小板分离和荧光团加载方法，该方法可产生功能反应性血小板，在其中我们能够使用基于微流控的筛选平台检测激动剂诱导的钙通量。血小板制备方案旨在尽量减少制备过程中诱导的血小板活化并优化信号强度。通过对荧光负载的人血小板中激动剂诱导的钙通量进行比率测定来监测血小板活化的测量，在宏观样品比色皿形式中进行了优化，以便为基于微流控芯片的检测做准备。对于这些研究中使用的微流控装置，每毫升1至2×10⁶个血小板的细胞密度和每秒5至10 nl的标称流速提供了最佳的事件分辨率，即每单位时间有5至20个血小板穿过检测体积。血小板对二磷酸腺苷和蛋白酶激活肽（PAR）1凝血酶受体激活肽（TRAP）呈剂量依赖性反应。此处展示的工作构成了原理验证实验，证明了微流控装置在针对人类血小板靶点进行高通量信号研究和药物发现筛选方面的应用潜力。
