使用新型 96 孔流量插件对 GPR68 进行定量机械刺激。

IF 5.4 2区 工程技术 Q1 BIOCHEMICAL RESEARCH METHODS
Lab on a Chip Pub Date : 2024-01-30 DOI:10.1039/D3LC00767G
Philipp Segeritz, Kirill Kolesnik, Daniel J. Scott and David J. Collins
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引用次数: 0

摘要

机械敏感蛋白在听觉、触觉和血流调节等一系列生理过程中发挥着至关重要的作用。虽然以前的工作已经证明了几种蛋白质的机械敏感性,但以一致、可复制的方式对细胞施加精确定义的机械力仍然是一项重大挑战。在这项工作中,我们提出了一种新型的 96 孔板兼容插件设备,用于生成高度可控的基于流的细胞机械模拟,从而实现对机械敏感性蛋白质功能的定量评估。该装置用于对表达机械敏感蛋白 GPR68(一种 G 蛋白偶联受体)的 HEK 293T 细胞进行机械刺激。通过测定流动细胞刺激过程中细胞内的钙水平,我们确定 GPR68 信号是外加剪切力的函数。由于这种方法与传统的细胞培养板兼容,并可在传统的荧光平板阅读器中同时读出,因此是研究机械传导的一种有价值的新工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Quantitative mechanical stimulation of GPR68 using a novel 96 well flow plugin†

Quantitative mechanical stimulation of GPR68 using a novel 96 well flow plugin†

Quantitative mechanical stimulation of GPR68 using a novel 96 well flow plugin†

Mechanosensitive proteins play a crucial role in a range of physiological processes, including hearing, tactile sensation and regulating blood flow. While previous work has demonstrated the mechanosensitivity of several proteins, the ability to apply precisely defined mechanical forces to cells in a consistent, replicable manner remains a significant challenge. In this work we present a novel 96-well plate-compatible plugin device for generating highly-controlled flow-based mechanical simulation of cells, which enables quantitative assessment of mechanosensitive protein function. The device is used to mechanically stimulate HEK 293T cells expressing the mechanosensitive protein GPR68, a G protein-coupled receptor. By assaying intracellular calcium levels during flow-based cell stimulation, we determine that GPR68 signalling is a function of the applied shear-force. As this approach is compatible with conventional cell culture plates and allows for simultaneous readout in a conventional fluorescence plate reader, this represents a valuable new tool to investigate mechanotransduction.

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来源期刊
Lab on a Chip
Lab on a Chip 工程技术-化学综合
CiteScore
11.10
自引率
8.20%
发文量
434
审稿时长
2.6 months
期刊介绍: Lab on a Chip is the premiere journal that publishes cutting-edge research in the field of miniaturization. By their very nature, microfluidic/nanofluidic/miniaturized systems are at the intersection of disciplines, spanning fundamental research to high-end application, which is reflected by the broad readership of the journal. Lab on a Chip publishes two types of papers on original research: full-length research papers and communications. Papers should demonstrate innovations, which can come from technical advancements or applications addressing pressing needs in globally important areas. The journal also publishes Comments, Reviews, and Perspectives.
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