Using FRET-Based Biosensor Cells to Study the Seeding Activity of Tau and α-Synuclein.
Using FRET-Based Biosensor Cells to Study the Seeding Activity of Tau and α-Synuclein.
复制标题
使用基于 FRET 的生物传感器细胞研究 Tau 和 α-突触核蛋白的接种活性。
DOI：
10.1007/978-1-0716-2597-2_10
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Bitan,Gal
中科院分区：
文献类型：
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作者：
Maina,KatherineN;Smet-Nocca,Caroline;Bitan,Gal
戴蒙德集团(德克萨斯大学西南分校)几年前开发的两个基于荧光共振能量转移(FRET)的生物传感器细胞系可以方便、灵敏和特异地测量在添加来自各种来源的tau和α-突触核蛋白的寡聚体或小聚集体“种子”后这些蛋白质在细胞内的聚集，这是相对于类似的单一荧光团系统的进步。这些生物传感器细胞系允许研究人员可视化tau或α-突触核蛋白的细胞内聚集，并使用流式细胞仪中的FRET信号以高灵敏度测量细胞内聚集。在这里，我们提供了产生种子、培养生物传感器细胞、用流式细胞术测量细胞内聚集物的详细方案，并对结果进行了分析，并讨论了该技术的实用性，目的是表征参与调节细胞内tau和α-突触核蛋白聚集的因素。
Two fluorescence resonance energy transfer (FRET)-based biosensor cell lines developed several years ago by the Diamond group (University of Texas, Southwestern) have allowed convenient, sensitive, and specific measurement of the intracellular aggregation of tau and α-synuclein following the addition of oligomer or small-aggregate “seeds” of these proteins from various sources, and an advancement relative to similar single-fluorophore systems. These biosensor cell lines allow researchers to both visualize the intracellular aggregates of tau or α-synuclein and measure intracellular aggregation with high sensitivity using a FRET signal in flow cytometry. Here we provide detailed protocols for generating seeds, culturing the biosensor cells, measuring intracellular aggregates by flow cytometry, and analyzing the results and discuss the utility of the technique with the aim of characterizing factors involved in the regulation of intracellular tau and α-synuclein aggregation.