title: Resolving the titer of murine cytomegalovirus by plaque assay using the M2-10B4 cell line and a low viscosity overlay.
abstract: BACKGROUND
  Murine cytomegalovirus (MCMV) is increasingly used as an infectious model to investigate host-pathogen interactions in mice. Detailed methods have been published for using primary murine embryonic fibroblasts (MEFs) for preparing stocks and determining viral titers of MCMV. For determining the titer of MCMV by plaque assay, these methods rely on a high viscosity media that restricts viral spreading through the supernatant of the culture, but is also usually too viscous to pipet. Moreover, MEFs must be repeatedly generated and can vary widely from batch-to-batch in purity, proliferation rates, and the development of senescence. In contrast, the M2-10B4 bone marrow stromal cell line (ATCC # CRL-1972), which is also permissive for MCMV, has been reported to produce high-titer stocks of MCMV and has the considerable advantages of growing rapidly and consistently. However, detailed methods using these cells have not been published.
  
  
  METHODS
  We modified existing protocols to use M2-10B4 cells for measuring MCMV titers by plaque assay.
  
  
  RESULTS
  We found that MCMV plaques could be easily resolved on monolayers of M2-10B4 cells. Moreover, plaques formed normally even when cultures of M2-10B4 cells were less than 50% confluent on the day of infection, as long as we also used a reduced viscosity overlay.
  
  
  CONCLUSIONS
  Overall, our protocol enabled us to use a consistent cell line to assess viral titers, rather than repeatedly producing primary MEFs. It also allowed us to start the assay with 4-fold fewer cells than would be required to generate a confluent monolayer, reducing the lead-time prior to the start of the assay. Finally, the reduced viscosity CMC could be handled by pipet and did not need to be pre-mixed with media, thus increasing its shelf-life and ease-of-use. We describe our results here, along with detailed protocols for the use of the M2-10B4 cell lines to determine the titer and grow stocks of MCMV.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Zurbach
authors.forename: Toktam
authors.initials: T
authors.lastname: Moghbeli
authors.affiliation: Department of Microbiology and Immunology, Jefferson Medical College, Kimmel Cancer Center, Thomas Jefferson University, 233 S, 10th St BLSB, rm 526, Philadelphia, PA 19107, USA. christopher.snyder@jefferson.edu.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Snyder
mesh_terms: D000818:Animals; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry*; D051379:Mice; D018146:Muromegalovirus / Q000302:isolation & purification*; D019562:Viral Load / Q000379:methods*; D010948:Viral Plaque Assay / Q000379:methods*; D014783:Viscosity
chemical_list: D003470:Culture Media
references.citation: PLoS Pathog. 2011 Oct;7(10):e1002295
references.pmid: 21998590
references.citation: Antimicrob Agents Chemother. 1985 Aug;28(2):240-5
references.pmid: 3010840
references.citation: J Virol. 2000 Nov;74(22):10729-36
references.pmid: 11044117
references.citation: J Immunol. 2009 Sep 15;183(6):3932-41
references.pmid: 19692644
references.citation: J Virol. 2011 Oct;85(19):10346-53
references.pmid: 21813614
references.citation: J Virol. 2012 Dec;86(23):12879-90
references.pmid: 22993151
references.citation: Curr Protoc Immunol. 2001 Aug;Chapter 19:Unit 19.7
references.pmid: 18432758
references.citation: J Virol. 2003 May;77(10):5557-70
references.pmid: 12719548
references.citation: J Clin Invest. 1982 Oct;70(4):762-8
references.pmid: 6288769
references.citation: J Exp Med. 1995 Oct 1;182(4):1045-56
references.pmid: 7561678
references.citation: J Virol. 2005 Aug;79(15):9492-502
references.pmid: 16014912
references.citation: J Virol. 1995 Feb;69(2):904-9
references.pmid: 7815559
references.citation: J Clin Invest. 1980 Apr;65(4):798-803
references.pmid: 6244333
references.citation: Virology. 1997 Jul 7;233(2):358-63
references.pmid: 9217058
references.citation: Arch Virol. 1989;107(3-4):253-60
references.pmid: 2554856
references.citation: Exp Hematol. 1988 Sep;16(8):718-26
references.pmid: 3261251
references.citation: Cancer Res. 1988 Nov 15;48(22):6438-43
references.pmid: 3263188
references.citation: Virol J. 2006 Aug 31;3:63
references.pmid: 16945126
references.citation: J Virol. 1985 Aug;55(2):264-73
references.pmid: 2991554
references.citation: J Virol Methods. 1997 Nov;68(2):193-8
references.pmid: 9389409
references.citation: J Virol. 1999 Aug;73(8):7056-60
references.pmid: 10400809