Analysis of Viral Infection and Viral and Cellular DNA and Proteins by Flow Cytometry

John M. Lehman1, Thomas D. Friedrich1, Judith Laffin1

1 Albany Medical College, Albany, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.17
DOI:  10.1002/0471142956.cy0717s17
Online Posting Date:  August, 2001
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Viruses are obligate intracellular parasites that require the host cell replication, transcription, and translation machinery for reproduction. Each viral group provides a unique series of viral‐cellular interactions. Studies have provided insight not only into viral replication and control of host functions, but also into cellular functions such as eukaryotic replication, transcription, and translation as well as the regulation of these events. This unit presents a protocol for flow cytometric monitoring of viral infection and quantitating viral‐cellular events. The availability of monoclonal and/or polyclonal antibodies directed to both viral and cellular proteins offers the ability to assay a specific molecule in the intact fixed cell and the opportunity to correlate viral events with cellular processes such as progression through the cell cycle.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Cells of interest, monolayer‐grown or suspension‐grown
  • PBS (see recipe)
  • 10× trypsin‐EDTA (Life Technologies), 37°C
  • Wash solution (see recipe)
  • Methanol, ice cold
  • Primary antibody (see recipe)
  • Secondary antibody (see recipe)
  • RNase A solution (see recipe)
  • Propidium iodide (PI; see recipe)
  • Inverted microscope to view monolayer cells and the trypsin‐EDTA dispersion
  • 37°C, 5% CO 2 incubator
  • 1.0‐ and 1.5‐ml microcentrifuge tubes
  • Variable microcentrifuge (e.g., Fisher Model 95A)
  • ∼50‐µm pore size nylon mesh (Nytex)
  • Flow cytometer with 488‐nm excitation and filters for collection of green (535‐nm band‐pass filter) and red (640‐nm long‐pass filter) fluorescence
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Literature Cited

   Cole, C. 1996. Polyomavirinae: The viruses and their replication. In Fundamental Virology, 3rd ed. (B.W. Fields, D.M. Knipe, and P.M. Howley, eds.) pp. 917‐945. Lippencott‐Raven Press, Philadelphia.
   Elmendorf, S., McSharry, J., Laffin, J., Fogleman, D., and Lehman, J.M. 1988. Detection of an early cytomegalovirus antigen with two‐color quantitative flow cytometry. Cytometry 9:254‐260.
   Friedrich, T.D., Laffin, J., and Lehman, J.M. 1993. Hypophosphorylated retinoblastoma gene product accumulates in SV40 infected CV‐1 cells acquiring a tetraploid DNA content. Oncogene 8:1673‐1677.
   Friedrich, T.D., Laffin, J., and Lehman, J.M. 1994. Induction of tetraploid DNA content by simian virus 40 is dependent on a T antigen function in G2 phase of the cell cycle. J. Virol. 68:4028‐4030.
   Jacobberger, J.W., Fogleman, D., and Lehman, J.M. 1986. Analysis of intracellular antigens by flow cytometry. Cytometry 7:356‐364.
   Laffin, J. and Lehman, J.M. 1994. Detection of intracellular virus and viral product. Methods Cell Biol. 41:543‐557.
   Lehman, J.M., Laffin, J., Jacobberger, J., and Fogleman, D. 1988. Analysis of simian virus 40 infection of permissive CV‐1 cells by quantitative two‐color fluorescence with flow cytometry. Cytometry 9:52‐59.
   Lehman, J.M., Friedrich, T.D., and Laffin, J. 1993. Quantitation of simian virus 40 T antigen correlated with the cell cycle of permissive and non‐permissive cells. Cytometry 14:401‐410.
   Lehman, J.M., Laffin, J., and Friedrich, T.D. 2000. Simian virus 40 induces multiple S phases with the majority of viral DNA replication in the G2 and second S phase in CV‐1 cells. Exp. Cell Res. 258:215‐222.
   McSharry, J.J., Costantino, R., Robbiano, E., Echols, R.M., Stevens, R., and Lehman, J.M. 1990. Detection and quantitation of human immunodeficiency virus‐infected peripheral blood mononuclear cells by flow cytometry. J. Clin. Microbiol. 28:724‐733.
   Whalen, B., Laffin, J., Friedrich, T.D., and Lehman, J.M. 1999. SV40 small t antigen enhances rereplication and alters expression of G1 regulatory proteins in permissive CV1 cells. Exp. Cell Res. 251:121‐127.
Key References
   Jacobberger et al., 1986. See above.
  Initial description of the protocol for viral protein and DNA detection.
   Laffin and Lehman, 1994. See above.
  A review of the protocol with specific comments addressing the steps and potential problems.
Internet Resources
  Information on general virology.
  Web page for The American Society for Microbiology (Virology).
  Web site for the International Society for Analytical Cytology.
  Web site for the American Type Culture Collection: cells, viruses, and hybridomas.‐1.htm
  Site providing Biosafety in Microbiological and Biomedical Laboratories publication.
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