| Multicolor Stimulated Emission Depletion (STED) Microscopy to Generate High-resolution Images of Respiratory Syncytial Virus Particles and Infected Cells
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Author:
Date:
2017-09-05
[Abstract] Human respiratory syncytial virus (RSV) infection in human lung epithelial A549 cells induces filopodia, cellular protrusions consisting of F-actin, that extend to neighboring uninfected cells (Mehedi et al., 2016). High-resolution imaging via stimulated emission depletion (STED) microscopy revealed filamentous RSV particles along these filopodia, suggesting that filopodia facilitate RSV cell-to-cell spread (Mehedi et al., 2016). In this protocol, we describe how to fix, permeabilize, immunostain, and mount RSV-infected A549 cells for STED imaging. We show that STED ...
[摘要] 人肺上皮A549细胞中的呼吸道合胞病毒（RSV）感染诱导丝状伪足，由F-肌动蛋白组成的细胞突起，延伸至相邻的未感染细胞（Mehedi等，2016）。 通过受激发射耗尽（STED）显微镜的高分辨率成像显示沿着这些丝状伪足的丝状RSV颗粒，表明丝状伪足有助于RSV细胞对细胞的扩散（Mehedi等，2016）。 在本协议中，我们描述如何修复，渗透，免疫染色和挂载RSV感染的A549细胞进行STED成像。 我们显示与共聚焦显微镜相比，STED增加了分辨率，可以通过使用去卷积软件的图像处理进一步改进。
【背景】RSV形成多形性病毒颗粒，其长度大约为直径约100nm，长度大约为10μm（Bachi和Howe，1973; Mehedi等，2016）。高分辨率光学显微技术是可视化RSV感染细胞和病毒颗粒之间相互作用的关键。在最近的一项研究中，我们使用超分辨率荧光显微镜来研究人肺上皮A549细胞中的RSV细胞对细胞的扩散。
STED显微镜是超分辨率显微镜技术之一，已被开发以规避约200nm衍射屏障的光限制（Hell和Wichmann，1994; Westphal等人，2008）。 ...
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| Human Astrovirus Propagation, Purification and Quantification
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Author:
Date:
2014-03-20
[Abstract] Astrovirus are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. Here, we describe astrovirus propagation, purification and titration. The Caco-2 human intestinal adenocarcinoma cell line is most widely used for studying astrovirus, although other cell lines, such as 293, T84 and LLC-MK2 can be used for propagation. However, Caco2 cells are desirable for their ability to form a differentiated intestinal epithelium, mimicking the human intestine and providing a realistic model for astrovirus growth and propagation.
[摘要] 星状病毒是小的，未发育的单链RNA病毒，其在多种哺乳动物和鸟类中引起腹泻。 在这里，我们描述星状病毒繁殖，纯化和滴定。 Caco-2人肠腺癌细胞系最广泛地用于研究星形病毒，尽管其它细胞系，例如293，T84和LLC-MK2可用于繁殖。 然而，Caco2细胞是其形成分化的肠上皮的能力，模拟人的肠并且为星状病毒生长和繁殖提供现实模型所需的。
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