Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells
Summary: 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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722005767 |
| _version_ | 1811292659376979968 |
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| author | Linda E. Franken Rene Rosch Ulrike Laugks Kay Grünewald |
| author_facet | Linda E. Franken Rene Rosch Ulrike Laugks Kay Grünewald |
| author_sort | Linda E. Franken |
| collection | DOAJ |
| description | Summary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-13T04:48:38Z |
| format | Article |
| id | doaj.art-526bc453dbf845bb9f68585bf3103030 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-13T04:48:38Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-526bc453dbf845bb9f68585bf31030302022-12-22T03:01:45ZengElsevierSTAR Protocols2666-16672022-12-0134101696Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cellsLinda E. Franken0Rene Rosch1Ulrike Laugks2Kay Grünewald3Leibniz Institute of Virology (LIV), Martinistraße 52, 20251 Hamburg, Germany; Centre for Structural Systems Biology, Notkestraße 85, 22607 Hamburg, Germany; Corresponding authorLeibniz 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, GermanyCentre for Structural Systems Biology, Notkestraße 85, 22607 Hamburg, Germany; Universität Hamburg, Institute for Biochemistry and Molecular Biology, Martin-Luther-King-Platz 6, 20146 Hamburg, GermanyLeibniz 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; Corresponding authorSummary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166722005767Cell biologyMicroscopyMolecular biologyStructural biologyCryo-EM |
| spellingShingle | Linda E. Franken Rene Rosch Ulrike Laugks Kay Grünewald Protocol for live-cell fluorescence-guided cryoFIB-milling and electron cryo-tomography of virus-infected cells STAR Protocols Cell biology Microscopy Molecular biology Structural biology Cryo-EM |
| 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 | Cell biology Microscopy Molecular biology Structural biology Cryo-EM |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722005767 |
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