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Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines
Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell p...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MyJove Corporation
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308597/ https://www.ncbi.nlm.nih.gov/pubmed/22090023 http://dx.doi.org/10.3791/3321 |
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author | Hui-Yuen, Joyce McAllister, Shane Koganti, Siva Hill, Erik Bhaduri-McIntosh, Sumita |
author_facet | Hui-Yuen, Joyce McAllister, Shane Koganti, Siva Hill, Erik Bhaduri-McIntosh, Sumita |
author_sort | Hui-Yuen, Joyce |
collection | PubMed |
description | Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis(1). LCL have been used to present antigens in a variety of immunologic assays(2, 3). In addition, LCL can be used to generate human monoclonal antibodies(4, 5) and provide a potentially unlimited source when access to primary biologic materials is limited(6, 7). A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide(8), and pokeweed mitogen(9) to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells(7, 10-12). The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23(hi)CD58(+) cells observed as early as three days post-infection indicates a successful outcome. |
format | Online Article Text |
id | pubmed-3308597 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | MyJove Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-33085972012-06-28 Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines Hui-Yuen, Joyce McAllister, Shane Koganti, Siva Hill, Erik Bhaduri-McIntosh, Sumita J Vis Exp Immunology Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis(1). LCL have been used to present antigens in a variety of immunologic assays(2, 3). In addition, LCL can be used to generate human monoclonal antibodies(4, 5) and provide a potentially unlimited source when access to primary biologic materials is limited(6, 7). A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide(8), and pokeweed mitogen(9) to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells(7, 10-12). The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23(hi)CD58(+) cells observed as early as three days post-infection indicates a successful outcome. MyJove Corporation 2011-11-08 /pmc/articles/PMC3308597/ /pubmed/22090023 http://dx.doi.org/10.3791/3321 Text en Copyright © 2011, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/ |
spellingShingle | Immunology Hui-Yuen, Joyce McAllister, Shane Koganti, Siva Hill, Erik Bhaduri-McIntosh, Sumita Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines |
title | Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines |
title_full | Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines |
title_fullStr | Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines |
title_full_unstemmed | Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines |
title_short | Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines |
title_sort | establishment of epstein-barr virus growth-transformed lymphoblastoid cell lines |
topic | Immunology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308597/ https://www.ncbi.nlm.nih.gov/pubmed/22090023 http://dx.doi.org/10.3791/3321 |
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