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

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-...

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Detalles Bibliográficos
Autores principales: Franken, Linda E., Rosch, Rene, Laugks, Ulrike, Grünewald, Kay
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9508610/
https://www.ncbi.nlm.nih.gov/pubmed/36149798
http://dx.doi.org/10.1016/j.xpro.2022.101696
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author Franken, Linda E.
Rosch, Rene
Laugks, Ulrike
Grünewald, Kay
author_facet Franken, Linda E.
Rosch, Rene
Laugks, Ulrike
Grünewald, Kay
author_sort Franken, Linda E.
collection PubMed
description 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).
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spelling pubmed-95086102022-09-25 Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells Franken, Linda E. Rosch, Rene Laugks, Ulrike Grünewald, Kay STAR Protoc Protocol 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). Elsevier 2022-09-22 /pmc/articles/PMC9508610/ /pubmed/36149798 http://dx.doi.org/10.1016/j.xpro.2022.101696 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Franken, Linda E.
Rosch, Rene
Laugks, Ulrike
Grünewald, Kay
Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells
title Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells
title_full Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells
title_fullStr Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells
title_full_unstemmed Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells
title_short Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells
title_sort protocol for live-cell fluorescence-guided cryofib-milling and electron cryo-tomography of virus-infected cells
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9508610/
https://www.ncbi.nlm.nih.gov/pubmed/36149798
http://dx.doi.org/10.1016/j.xpro.2022.101696
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