Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.
Université de Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1167 - RID-AGE - Risk Factors and Molecular Determinants of Aging-Related Diseases, Lille, France.
Methods Mol Biol. 2023;2551:125-145. doi: 10.1007/978-1-0716-2597-2_10.
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.
几年前，由 Diamond 小组（德克萨斯大学西南医学中心）开发的两种基于荧光共振能量转移（FRET）的生物传感器细胞系，允许方便、敏感和特异性地测量在添加来自各种来源的这些蛋白质的寡聚体或小聚集体“种子”后，tau 和 α-突触核蛋白在细胞内的聚集，这是相对于类似的单荧光团系统的一项进展。这些生物传感器细胞系允许研究人员使用流式细胞术的 FRET 信号既可视化 tau 或 α-突触核蛋白的细胞内聚集体，又以高灵敏度测量细胞内聚集。在这里，我们提供了详细的方案来生成种子、培养生物传感器细胞、通过流式细胞术测量细胞内聚集体，并分析结果，讨论该技术的实用性，旨在表征参与调节细胞内 tau 和 α-突触核蛋白聚集的因素。
