Establishment of Permanent Cell Lines by Epstein‐Barr Virus Transformation

John Gilbert1

1 Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3H
DOI:  10.1002/0471142905.hga03hs02
Online Posting Date:  May, 2001
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Abstract

This basic protocol presents Epstein‐Barr virus (EBV) transformation of B cells to create a permanent cell line to provide cells for genetic and DNA analysis. Virus stocks are prepared from cultures of an infected marmoset cell line in the first support protocol. Transformed cells may be analyzed immediately or frozen for future use as described in the second support protocol.

     
 
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Table of Contents

  • Basic Protocol 1: Epstein‐Barr Virus Transformation of Cultured Lymphocytes
  • Support Protocol 1: Preparation of Epstein‐Barr Virus Stock
  • Support Protocol 2: Freezing and Reculturing of Epstein‐Barr Virus–Transformed Lymphocytes
  • Reagents and Solutions
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Epstein‐Barr Virus Transformation of Cultured Lymphocytes

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • Whole blood collected in 8.5‐ml yellow‐top Vacutainer tubes with ACD solution A (Becton Dickinson Labware)
  • HBSS ( appendix 2) without Ca2+ or Mg2+
  • recipeTransformation medium (see recipe), 37°C
  • recipeGrowth medium (see recipe), 37°C
  • Leuco‐prep tubes (Becton Dickinson Labware)
  • 15‐ml conical centrifuge tube, sterile
  • 25‐ and 75‐cm2 tissue culture flasks
  • Additional reagents and equipment for mammalian tissue culture ( appendix 3G)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Preparation of Epstein‐Barr Virus Stock

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • EBV‐infected B95‐8 cells (ATCC)
  • Complete Iscoves modified Dulbeccos medium (IMDM; e.g., Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS), 37°C
  • 25‐ and 75‐cm2 tissue culture flasks
  • 50‐ml centrifuge tubes
  • 95% (v/v) dry ice/ethanol bath or liquid nitrogen
  • 0.45‐µm filter
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Freezing and Reculturing of Epstein‐Barr Virus–Transformed Lymphocytes

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • Cultures of EBV‐transformed lymphocytes ( protocol 1)
  • recipeFreezing medium (see recipe)
  • Complete RPMI, serum‐free ( appendix 3G)
  • 50‐ml centrifuge tubes
  • 15‐ml centrifuge tubes
  • 1.8‐ml cryovial (USA/Scientific Plastics)
  • Additional reagents and equipment to determine cell viability by trypan blue exclusion ( appendix 3G)
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Figures

Videos

Literature Cited

Literature Cited
   Miller, G. and Lipman, M. 1973. Release of infectious Epstein‐Barr virus by transformed marmoset leucocytes. Proc. Natl. Acad. Sci. U.S.A. 70:190‐194.
   Tosato, G., Pike, S.E., Koski, L.E., and Blease, R.M. 1982. Selective inhibition of immunoregulatory cell functions by Cyclosporin A. J. Immunol. 128:1986‐1991.
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