Diagnosis and Treatment of Mycoplasma‐Contaminated Cell Cultures

Helena Harlin1, Thomas F. Gajewski1

1 University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Cytometry
Unit Number:  Appendix 3C
DOI:  10.1002/0471142956.cya03cs43
Online Posting Date:  January, 2008
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Abstract

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures. Curr. Protocol. Cytom. 43:A.3C.1‐A.3C.7. © 2008 by John Wiley & Sons, Inc.

Keywords: mycoplasma; PCR; adenosine phosphorylase; ciprofloxacin; BM‐cyclin

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Testing for Mycoplasma Infection by PCR
  • Alternate Protocol 1: Testing for Mycoplasma Infection by Detection of Adenosine Phosphorylase Secretion
  • Basic Protocol 2: Treatment of Mycoplasma‐Contaminated Cell Cultures with Ciprofloxacin
  • Alternate Protocol 2: Treatment of Mycoplasma‐Contaminated Cell Cultures with BM‐Cyclin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Testing for Mycoplasma Infection by PCR

  Materials
  • Cell cultures to be tested for contamination
  • Phosphate‐buffered saline (PBS; appendix 2A) or culture medium for cells
  • H 2O, sterile
  • Roche Mycoplasma PCR ELISA kit (cat. no. 1663925) including:
    • Lysis reagent
    • Mycoplasma‐positive control DNA
    • Neutralization reagent
    • PCR ready‐to‐go mix
  • Taq DNA polymerase
  • Thin‐walled PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (Sambrook and Russell, ; Voytas, )

Alternate Protocol 1: Testing for Mycoplasma Infection by Detection of Adenosine Phosphorylase Secretion

  Materials
  • SP2/0‐Ag14 hybridoma cells (ATCC #CRL 1581)
  • Cells to be tested in appropriate medium without antibiotics
  • MycoTect (Life Technologies) containing 6‐methylpurine deoxyriboside (6‐MPDR)
  • 6‐methylpurine (6‐MP; Sigma)
  • 96‐well flat‐bottom microtiter plates (Costar, Falcon, or equivalent)

Basic Protocol 2: Treatment of Mycoplasma‐Contaminated Cell Cultures with Ciprofloxacin

  Materials
  • 10 mg/ml ciprofloxacin⋅HCl (see recipe), sterile
  • Contaminated cells (see protocol 1 and protocol 2)

Alternate Protocol 2: Treatment of Mycoplasma‐Contaminated Cell Cultures with BM‐Cyclin

  Materials
  • BM‐cyclin solutions 1 and 2 (see recipe), sterile
  • Contaminated cells (see protocol 1 and protocol 2)
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Figures

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Literature Cited

   McGarrity, G.J. and Carson, D.A. 1982. Adenosine phosphorylase‐mediated nucleoside toxicity: Application towards the detection of mycoplasmal infection in mammalian cell cultures. Exp. Cell Res. 139:199‐205.
   Phelan, M. 2006. Techniques for mammalian cell culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Sambrook, J. and Russell, D. 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Schmitt, K., Däubeaner, W., Bitter‐Suermann, D., and Hadding, U. 1988. A safe and efficient method for elimination of cell culture mycoplasmas using ciprofloxacin. J. Immunol. Methods 109:17‐25.
   Shulman, M., Wilde, C.D., and Köhler, G. 1978. A better cell line for making hybridomas secreting specific antibodies. Nature (Lond.) 276:269‐270.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Wirth, M., Grashoff, M., Schumacher, L., and Hauser, H. 1995. Mycoplasma detection by the Mycoplasma PCR ELISA. Biochemica 3:33‐35.
   Yokoyama, W., Christensen, M., Dos Santos, G., and Miller, D. 2006. Production of monoclonal antibodies. Curr. Protoc. Immunol. 74:2.5.1‐2.5.25.
Key Reference
   McGarrity, G.J., Sarama, J., and Vanaman, V. 1979. Factors influencing microbiological assay of cell‐culture Mycoplasmas. In Vitro (Rockville) 15:73‐81.
  Classic review of mycoplasma contamination and its evaluation.
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