Abstract
BackgroundMurine 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.MethodsWe modified existing protocols to use M2-10B4 cells for measuring MCMV titers by plaque assay.ResultsWe 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.ConclusionsOverall, 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.
Highlights
Murine cytomegalovirus (MCMV) is increasingly used as an infectious model to investigate host-pathogen interactions in mice
We developed a robust protocol for using M2-10B4 cells that requires fewer cells to start the assay and a lower viscosity overlay than the previously published protocols
MCMV was serially diluted in media in 10-fold increments and 100 μl of diluted virus added to 1 mL of media in each well
Summary
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. Murine cytomegalovirus (MCMV) is increasingly used as an infectious model to investigate cytomegalovirus (CMV) biology, CMV-driven immune responses and host-pathogen interactions. Murine embryonic fibroblasts (MEFs) are the “gold standard” cell type for the growth of MCMV [1]. These cells are not immortalized and must be produced repeatedly from freshly harvested mouse embryos. If new mice were needed to generate more MEFs, the delay could last several weeks or more
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