Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection
Summary: Connexins (Cxs) play a crucial role in maintaining lens transparency. Here, we present a protocol for altering Cx hemichannel (HC) function in primary chicken lens fiber cells using high-titer retroviral replication competent avian sarcoma-leukosis virus long terminal repeat with splice acc...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723005312 |
| _version_ | 1797677652657569792 |
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| author | Yu Du Francisca M. Acosta Jianping Zhang Yuxin Tong Yumeng Quan Sumin Gu Jean X. Jiang |
| author_facet | Yu Du Francisca M. Acosta Jianping Zhang Yuxin Tong Yumeng Quan Sumin Gu Jean X. Jiang |
| author_sort | Yu Du |
| collection | DOAJ |
| description | Summary: Connexins (Cxs) play a crucial role in maintaining lens transparency. Here, we present a protocol for altering Cx hemichannel (HC) function in primary chicken lens fiber cells using high-titer retroviral replication competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor (A) infection. We describe steps for incubating eggs, isolating lenses, culturing cells, preparing reagents, and infecting cells. We then detail cell treatment and detection of apoptosis and death. This protocol can assess protein kinase A, HC activity, and increased glutathione transport for protecting lens fiber cells against oxidative stress.For complete details on the use and execution of this protocol, please refer to Liu et al.,1 Riquelme et al.,2 Shi et al.,3 Jiang,4 and Rath et al.5 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-03-11T22:48:23Z |
| format | Article |
| id | doaj.art-015f1d15c41a4152a77ce377a62a05f0 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-03-11T22:48:23Z |
| publishDate | 2023-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-015f1d15c41a4152a77ce377a62a05f02023-09-22T04:39:40ZengElsevierSTAR Protocols2666-16672023-12-0144102564Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infectionYu Du0Francisca M. Acosta1Jianping Zhang2Yuxin Tong3Yumeng Quan4Sumin Gu5Jean X. Jiang6Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA; Department of Ophthalmology, Lanzhou University Second Hospital; Second Clinical School, Lanzhou University, Lanzhou, Gansu 730000, China; Corresponding authorDepartment of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USADepartment of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USADepartment of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USADepartment of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USADepartment of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USADepartment of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA; Corresponding authorSummary: Connexins (Cxs) play a crucial role in maintaining lens transparency. Here, we present a protocol for altering Cx hemichannel (HC) function in primary chicken lens fiber cells using high-titer retroviral replication competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor (A) infection. We describe steps for incubating eggs, isolating lenses, culturing cells, preparing reagents, and infecting cells. We then detail cell treatment and detection of apoptosis and death. This protocol can assess protein kinase A, HC activity, and increased glutathione transport for protecting lens fiber cells against oxidative stress.For complete details on the use and execution of this protocol, please refer to Liu et al.,1 Riquelme et al.,2 Shi et al.,3 Jiang,4 and Rath et al.5 : 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/S2666166723005312Cell BiologyCell-Based Assays |
| spellingShingle | Yu Du Francisca M. Acosta Jianping Zhang Yuxin Tong Yumeng Quan Sumin Gu Jean X. Jiang Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection STAR Protocols Cell Biology Cell-Based Assays |
| title | Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection |
| title_full | Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection |
| title_fullStr | Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection |
| title_full_unstemmed | Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection |
| title_short | Protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high-titer retroviral RCAS(A) infection |
| title_sort | protocol for altering connexin hemichannel function in primary chicken lens fiber cells using high titer retroviral rcas a infection |
| topic | Cell Biology Cell-Based Assays |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723005312 |
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