Rapid Titration of Viruses by Flow Cytometry

Nir Drayman1, Ariella Oppenheim1

1 Department of Hematology, Hadassah Medical School, The Hebrew University of Jerusalem, Jerusalem, Israel
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.11
DOI:  10.1002/0471143030.cb2611s51
Online Posting Date:  June, 2011
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Abstract

Traditionally, the most common methods used to titrate virus stocks are the plaque assay and the hemagglutination assay. The protocol presented here is based on the detection of viral‐expressed proteins in infected cells by flow cytometry. It is simpler and more rapid than the traditional plaque‐forming assay and it enables high‐throughput analyses. Curr. Protoc. Cell Biol. 51:26.11.1‐26.11.7. © 2011 by John Wiley & Sons, Inc.

Keywords: virus titration; FACS; SV40

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cells (e.g., CV1 cells for SV40 titration)
  • Cell culture medium
  • Viral stock (e.g., SV40)
  • Serum‐free medium (SFM; cell culture medium without serum)
  • Phosphate‐buffered saline (PBS; Biological Industries, cat. no. 02‐023‐1)
  • 0.05% EDTA in PBS
  • Trypsin solution, to detach cells from the plate (Biological Industries, cat. no. 03‐050‐1)
  • 100% Methanol (keep at −20°C)
  • Primary antibody in blocking solution (for SV40 titration: anti‐T antigen; Pab 101, Santa Cruz)
  • Blocking solution: 5% bovine serum albumin (BSA) in PBS
  • Fluoropohore‐conjugated secondary antibody
  • 6‐well plates
  • 37°C incubator
  • Hemacytometer
  • 1.5‐ml microcentrifuge tubes
  • Vortex
  • 15‐ml conical centrifuge tubes
  • Centrifuge
  • 53‐µm filter
  • FACS tubes
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Figures

Videos

Literature Cited

Literature Cited
   Drayman, N., Kler, S., Ben‐Nun‐Shaul, O., and Oppenheim, A. 2010. Rapid method for SV40 titration. J. Virol. Methods 164:145‐147.
   Gueret, V., Negrete‐Virgen, J.A., Lyddiatt, A., and Al‐Rubeai, M. 2002. Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells. Cytotechnology 38:87‐97.
   Li, Z., Ling, L., Liu, X., Laus, R., and Delcayre, A. 2010. A flow cytometry‐based immuno‐titration assay for rapid and accurate titer determination of modified vaccinia Ankara virus vectors. J. Virol. Methods 169:87‐94.
   Lonsdale, R., Pau, M.G., Oerlemans, M., Ophorst, C., Vooys, A., Havenga, M., Goudsmit, J., UytdeHaag, F., and Marzio, G. 2003. A rapid method for immunotitration of influenza viruses using flow cytometry. J. Virol. Methods 110:67‐71.
   Nair, A., Xie, J., Joshi, S., Harden, P., Davies, J., and Hermiston, T. 2008. A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors. J. Virol. Methods 153:269‐272.
   Thomas, M.A., Lichtenstein, D.L., Krajcsi, P., and Wold, W.S. 2007. A real‐time PCR method to rapidly titer adenovirus stocks. Methods Mol. Med. 130:185‐192.
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