Platelet Functional Testing Via High-Throughput Microtiter Plate-Based Assays.
Platelet Functional Testing Via High-Throughput Microtiter Plate-Based Assays.
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通过高通量微量滴定板检测进行血小板功能测试。
DOI：
10.1002/cpz1.668
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Tourdot,BenjaminE
中科院分区：
文献类型：
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作者：
Tamang,HemKumar;Stringham,EmilyN;Tourdot,BenjaminE
血小板在止血和血栓形成中发挥着关键作用；因此，测量血小板反应性的体外测定是深入了解这些生理过程、诊断血小板疾病和开发抗血栓疗法的基本工具。然而，传统的血小板测定（例如评估血小板功能的临床金标准）聚合度测定法的通量低，并且需要专门的设备。由于血小板在体内的寿命有限，因此小型化和多重检测的过程可以在比传统检测少得多的时间内对血小板功能进行更广泛的概述。几个小组已经开发出简化的高通量方法，使用标准实验室设备量化血小板活化，以降低研究血小板生物学的门槛。本文描述了一组经过优化和验证的高通量微孔板检测，可独立或组合地全面评估血小板功能，以提高通量并降低成本。具体来说，刺激血小板后，可以使用酶标仪通过吸光度测量光透射聚集；基于 ATP 依赖性发光产生的致密颗粒分泌；使用可渗透细胞的荧光 Ca2+ 敏感染料检测细胞质钙水平。此外，血小板是血液中易于获取的成分，与其他细胞共享信号通路，使其成为高通量药物筛选的理想选择。本文提出的高度适应性和互补性测定可用于破译血小板激活的分子机制或识别新型抑制剂。 © 2023 作者。 Wiley periodicals LLC 出版的当前方案。基本方案 1：基于微量滴定板的光透射聚集测量基本方案 2：在高通量微孔板测定中测量致密颗粒分泌基本方案 3：基于微量滴定板的钙动员支持方案：血小板分离和计数
Platelets play a critical role in hemostasis and thrombosis; therefore,in vitroassays that measure platelet reactivity are fundamental tools to gain insight into these physiologic processes, to diagnose platelet disorders, and to develop antithrombotic therapies. However, conventional platelet assays such as aggregometry, the clinical gold standard for assessing platelet function, are low throughput and require specialized equipment. Since platelets have a finite life spanex vivo, processes to miniaturize and multiplex assays allow a much broader overview of platelet function in significantly less time than conventional assays. Several groups have developed simplified, high‐throughput approaches to quantify platelet activation with standard laboratory equipment to lower the barrier of entry to study platelet biology. This article describes a panel of optimized and validated high‐throughput microplate assays to comprehensively assess platelet functionality, independently or in combination, to increase throughput and reduce costs. Specifically, following stimulation of platelets, a plate reader can be used to measure light transmission aggregation via absorbance; dense‐granule secretion based on ATP‐dependent luminescence generation; and cytosolic calcium levels with a cell‐permeant, fluorescent Ca2+‐sensitive dye. Additionally, platelets are an easily accessible component of the blood that share signaling pathways with other cells, making them ideal for high‐throughput drug screens. The highly adaptable and complementary assays presented in this article can be used to decipher the molecular mechanism underlying platelet activation or to identify novel inhibitors. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Microtiter plate–based light transmission aggregometryBasic Protocol 2: Measuring dense‐granule secretion in high‐throughput microplate assaysBasic Protocol 3: Microtiter plate–based calcium mobilizationSupport Protocol: Platelet isolation and enumeration