Generation of Epstein‐Barr Virus (EBV)–Immortalized B Cell Lines

Giovanna Tosato1, Jeffrey I. Cohen2

1 National Cancer Institute, Bethesda, Maryland, 2 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.22
DOI:  10.1002/0471142735.im0722s76
Online Posting Date:  February, 2007
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Immortalization of B lymphocytes by EBV is an effective procedure for inducing long‐term growth of certain human B lymphocytes. The protocol described in this unit to accomplish this can be divided into three stages: preparation of virus, preparation of target cells to be immortalized, and EBV infection and growth of infected cells.

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Table of Contents

  • Commentary
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Basic Protocol 1:

  • Complete RPMI‐10 medium ( appendix 22), without and with 1 µg/ml cyclosporin A (Sigma‐Aldrich, cat. no. C‐1832; prepare 1 mg/ml stock in 100% ethanol and store at −20°C)
  • EBV B95‐8 virus (ATCC #VR‐1492 or Coriell Institute for Medical Research ID EBV, or B95‐8 cells (marmoset cell line; no longer commercially available)
  • Heparinized peripheral blood ( appendix 3F)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Ficoll‐Paque (GE Healthcare Life Sciences)
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • Beckman centrifuge with JS 4.2 rotor (or equivalent)
  • 0.45‐µm filter
  • 50‐ml conical tubes
  • 25‐cm2 tissue culture flasks
  • Additional reagents and equipment for cell counting, cryopreservation, and determination of viability ( appendix 33) and Ficoll gradient centrifugation (unit 7.1)
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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.
   Chang, I.C., Wu, J.Y., Lu, H.I., Ko, H.W., Kuo, J.L., Wang, C.Y., Shen, P.S., and Hwang, S.M. 2006. High‐potentiality preliminary selection criteria and transformation time–dependent factors analysis for establishing Epstein‐Barr virus–transformed human lymphoblastoid cell lines. Cell Prolif. 39:457‐469.
   Elliott, J., Coulter‐Mackie, M.B., Jung, J.H., Rodenhiser, D.I., and Singh, S.M. 1991. A method of transforming lymphocytes from very small blood volumes suitable for pediatric samples. Hum. Genet. 86:615‐616.
   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.
   Neitzel, H. 1986. A routine method for establishment of permanent growing lymphoblastoid cell lines. Hum. Genet. 73:320‐326.
   Nilsson, K. and Klein, G. 1982. Phenotypic and cytogenetic characteristics of human B lymphoid cell lines and their relevance for the etiology of Burkitt's lymphoma. Adv. Cancer Res. 37:319‐380.
   Rickinson, A.B., Moss, D.J., and Pope, J.H. 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.
   Sugden, B. and Mark, W. 1977. Clonal transformation of adult human leukocytes by Epstein‐Barr virus. J. Virol. 23:503‐508.
   Sugden, B., Phelps, M., and Domoradski, J. 1979. Epstein‐Barr virus DNA is amplified in transformed lymphocytes. J. Virol. 31:590‐595.
   Sun, R., Grogan, E., Shedd, D., Bykovsky, A.F., Kushnaryov, V.M., Grossberg, S.E., and Miller, G. 1995. Transmissible retrovirus in Epstein‐Barr virus‐producer B95‐8 cells. Virology 209:374‐383.
   Tohda, H., Oikawa, A., Kudo, T., and Tachibana, T. 1978. A greatly simplified method of establishing B‐lymphoblastoid cell lines. Cancer Res. 38:3560‐3562.
   Tosato, G., Pike, S.E., Koski, I., and Blaese, R.M. 1982. Selective inhibition of immunoregulatory cell functions by Cyclosporin A. J. Immunol. 128:1986‐1991.
Key Reference
   Neitzel, 1986. See above.
  Presents a detailed description of human B lymphoid cell lines.
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