活细胞荧光引导的 cryoFIB 铣切和电子冷冻断层成像病毒感染细胞的方案。

Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells.

机构信息

Leibniz Institute of Virology (LIV), Martinistraße 52, 20251 Hamburg, Germany; Centre for Structural Systems Biology, Notkestraße 85, 22607 Hamburg, Germany.

Leibniz Institute of Virology (LIV), Martinistraße 52, 20251 Hamburg, Germany; Centre for Structural Systems Biology, Notkestraße 85, 22607 Hamburg, Germany; Universität Hamburg, Institute for Biochemistry and Molecular Biology, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.

出版信息

STAR Protoc. 2022 Dec 16;3(4):101696. doi: 10.1016/j.xpro.2022.101696. Epub 2022 Sep 22.

DOI:10.1016/j.xpro.2022.101696

PMID:36149798

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9508610/

Abstract

Here, we present a protocol for assessing virus-infected cells using electron cryo-tomography (cryoET). It includes the basic workflows of seeding cells, plunge-freezing, clipping, cryo-focused ion beam milling (cryoFIB-milling), and cryoET, as well as two optional modules: micropatterning and live-cell fluorescence microscopy. We use an A549 human cell line and the virus HAdV5-pIX-mcherry in this protocol, but the comprehensive workflow can be easily transferred to other cell types and different types of virus infection or treatment. For complete details on the use and execution of this protocol, please refer to Pfitzner et al. (2021).

摘要

在这里，我们展示了使用电子冷冻断层成像术（cryoET）评估病毒感染细胞的方案。它包括细胞接种、无浸泡冷冻、切割、冷冻聚焦离子束铣削（cryoFIB 铣削）和 cryoET 的基本工作流程，以及两个可选模块：微图案化和活细胞荧光显微镜。在本方案中，我们使用了 A549 人细胞系和病毒 HAdV5-pIX-mcherry，但全面的工作流程可以很容易地转移到其他细胞类型和不同类型的病毒感染或处理。有关该方案使用和执行的完整详细信息，请参阅 Pfitzner 等人（2021 年）。