Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors
Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors
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DOI：
10.1007/978-1-4939-8612-5_6
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发表时间：
2018-01-01
期刊：
影响因子：
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通讯作者：
Collins, Sean R.
中科院分区：
文献类型：
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作者：
Bell, George R. R.;Natwick, Dean E.;Collins, Sean R.
中性粒细胞是先天免疫反应中关键的早期反应者，它利用趋化性（沿着化学梯度定向迁移）到达感染或炎症部位。这一过程需要将细胞表面受体的输入与细胞的极性和运动信号网络整合起来，其中 Rho 家族 GTP 酶的高度动态且相互关联的信号发挥着核心作用。为了理解这一根本性的重要行为，我们描述了一种高分辨率、琼脂糖下趋化性测定，用于中性粒细胞样细胞系（HL-60 或 PLB-985）或原代中性粒细胞。我们还描述了如何在该测定中使用化学引诱剂的光学解笼来刺激细胞。这些技术与落射荧光、全内反射荧光 (TIRF) 和共焦显微镜兼容。此外，我们还介绍了如何使用基于福斯特共振能量转移 (FRET) 的生物传感器测量 Rho 家族 GTP 酶的活性。本章概述的具体实验步骤包括如何（1）设置琼脂糖下测定，（2）光学图案化学引诱剂梯度，（3）对细胞进行成像，以及（4）对 FRET 生物传感器进行基本图像分析。
Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Forster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.