Generation of Continuously Growing B Cell Lines by Epstein‐Barr Virus Transformation

William E. Biddison1

1 National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 2.4
DOI:  10.1002/0471143030.cb0204s01
Online Posting Date:  May, 2001
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Primary cultures of cells tend to have a limited life span, which in turn limits the availability of a consistent population of cells to study. In this unit, Epstein‐Barr virus produced by a marmoset cell line is used to transform B cells into a continuously growing cell line that produces no virus. These cell lines can be used as a constant source of cells for further study.

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Table of Contents

  • Reagents and Solutions
  • Commentary
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Basic Protocol 1:

  • Complete culture medium (see recipe), 37°C
  • B95‐8 EBV‐transformed marmoset cell line (ATCC #CRL 1612)
  • Anti‐CD3 monoclonal antibody produced by OKT3 hybridoma (ATCC #CRL 8001)
  • 25‐cm2 and 75‐mm2 tissue culture flasks
  • Sorvall RT‐6000B centrifuge with H‐1000 rotor (or equivalent refrigerated centrifuge and rotor) and 50‐ml centrifuge tubes
  • 0.45‐µm sterile filter
  • Additional reagents and equipment for growing cells, determining cell viability by trypan blue exclusion, and cryopreservation of cells (unit 1.1) and preparation of peripheral blood lymphocytes (unit 2.3)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.
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Literature Cited

Literature Cited
   Aman, P., Ehlin‐Henriksson, B., and Klein, G. 1984. Epstein‐Barr virus susceptibility of normal human B lymphocyte populations. J. Exp. Med. 159:208‐220.
   Falk, K., Rotzschke, O., Stevanovic, S., Jung, G., and Rammensee, H.‐G. 1991. Allele‐specific motifs revealed by sequencing of self‐peptides eluted from MHC molecules. Nature 351:290‐293.
   Miller, G. and Lipman, M. 1973. Release of infectious Epstein‐Barr virus by transformed marmoset leukocytes. Proc. Natl. Acad. Sci. U.S.A. 70:190‐194.
   Orr, H., Lopez de Castro, J., Parham, P., Ploegh, H., and Strominger, J. 1979. Comparison of amino acid sequences of two human histocompatibility antigens: HLA‐A2 and HLA‐B7: Location of putative alloantigenic sites. Proc. Natl. Acad. Sci. U.S.A. 76:4395‐4399.
   Rickinson, A., Moss, D., and Pope, J. 1979. Long‐term T cell–mediated immunity to Epstein‐Barr virus in man. II. Components necessary for regression in virus‐infected leukocyte cultures. Int. J. Cancer 23:610‐617.
   Roche, P. and Cresswell, P. 1990. Invariant chain association with HLA‐DR molecules inhibits immunogenic peptide binding. Nature 345:615‐618.
   Sugden, B. and Mark, W. 1977. Clonal transformation of adult human leukocytes by Epstein‐Barr virus. J. Virol. 23:503‐508.
   Tosato, G., Pike, S., Koski, I., and Blaese, R. 1982. Selective inhibition of immunoregulatory cell functions by Cyclosporin A. J. Immunol. 128:1986‐1991.
Key Reference
   Miller and Lipman 1973. See above.
  Presents detailed description of B cell transformation and determination of EBV titers using umbilical cord leukocytes.
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