Isolation and Immortalization of Lymphocytes

Paul D. Ling1, Helen M. Huls1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 28.2
DOI:  10.1002/0471142727.mb2802s70
Online Posting Date:  May, 2005
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Abstract

Recent advances in Genomics and Proteomics have necessitated a constant supply of DNA derived from specific genotypes. Lymphoblastoid cell lines (LCLs) are of great practical value as an unlimited source of stable genomic DNA and viable cells, which can be used to perform a variety of biochemical and molecular studies. LCLs are typically generated by infection of primary lymphocytes with Epstein‐Barr virus (EBV). In this unit, we describe simple procedures for production of EBV, isolation of lymphocytes, EBV infection of lymphocytes, and isolation of the resulting LCLs.

Keywords: B lymphocyte; Epstein‐Barr virus; Immortalization; Lymphoblastoid cell line (LCL)

     
 
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Table of Contents

  • Basic Protocol 1: Epstein‐Barr Virus–Mediated Immortalization of Human B Lymphocytes
  • Support Protocol 1: Titration of Virus on Lymphocytes
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Epstein‐Barr Virus–Mediated Immortalization of Human B Lymphocytes

  Materials
  • B95‐8 cells
  • Growth medium: RPMI‐1640 medium ( appendix 3F), containing 200 mM L‐glutamine, with or without fetal bovine serum (FBS; appendix 3F)
  • Ficoll‐Paque (Amersham Biosciences 17‐1440‐02) or Accuspin system‐histopaque‐1077 tubes (SIGMA diagnostics A 6929)
  • Anti‐coagulated human blood, freshly obtained
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Cyclosporine A (Sigma‐Aldrich)
  • Storage medium: RPMI medium containing 20% to 40% (v/v) FBS and 10% (v/v) DMSO
  • 15‐ and 50‐ml sterile plastic, screw‐cap, conical centrifuge tubes
  • 0.45‐µm filters
  • 96‐well flat‐welled tissue culture plates, sterile
  • 24‐well sterile tissue culture plates or T‐25 tissue culture flasks, sterile
  • Additional reagents and equipment for mammalian cell culture ( appendix 3F)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Titration of Virus on Lymphocytes

  Materials
  • Lymphocytes, Ficoll‐Paque purified ( protocol 1, steps to )
  • Culture medium: RPMI ( appendix 3F) containing 10% (v/v) FBS and 1 µg/ml cyclosporine A
  • B95‐8 virus stocks to be titered
  • 96‐well flat‐bottomed plates
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Figures

Videos

Literature Cited

   Bird, A.G., McLachlan, S.M., and Britton, S. 1981. Cyclosporin A promotes spontaneous outgrowth in vitro of Epstein‐Barr virus‐induced B‐cell lines. Nature 289:300‐301.
   Bornkamm, G.W. and Hammerschmidt, W. 2001. Molecular virology of Epstein‐Barr virus. Philos. Trans. R. Soc. Lond. B Biol. Sci. 356:437‐459.
   Epstein, M.A., Achong, B.G., and Barr, Y.M. 1964. Virus particles in cultured lymphocytes from Burkitt's lymphoma. Lancet 1:702‐703.
   Henle, W., Diehl, V., Kohn, G., Zur Hausen, H., and Henle, G. 1967. Herpes‐type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells. Science 157:1064‐1065.
   Miller, G., Shope, T., Lisco, H., Stitt, D., and Lipman, M. 1972. Epstein‐Barr virus: Transformation, cytopathic changes, and viral antigens in squirrel monkey and marmoset leukocytes. Proc. Natl. Acad. Sci. U.S.A. 69:383‐387.
   Neitzel, H. 1986. A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum. Genet. 73:320‐326.
   Rickinson, A.B. and Kieff, E. 2001. Epstein‐Barr Virus. 4th ed. In Virology (D.M. Knipe and P.M. Howley, Eds.), Vol. 2, pp. 2575‐2627. Lippincott‐Raven Publishers, Philadelphia.
   Sugden, B. and Mark, W. 1977. Clonal transformation of adult human leukocytes by Epstein‐Barr virus. J. Virol. 23:503‐508.
Internet Resources
   http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm
  Web site for acquiring the most recent CDC handbook, Biosafety in Microbiological and Biomedical laboratories (BMBL) 4th edition.
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