Resolving the titer of murine cytomegalovirus by plaque assay using the M2-10B4 cell line and a low viscosity overlay.
Resolving the titer of murine cytomegalovirus by plaque assay using the M2-10B4 cell line and a low viscosity overlay.
复制标题
通过使用M2-10B4细胞系和低粘度覆盖层来解决鼠巨细胞病毒的滴度。
DOI：
10.1186/1743-422x-11-71
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发表时间：
2014-04-18
期刊：
影响因子：
4.8
通讯作者：
Snyder CM
中科院分区：
文献类型：
--
作者：
Zurbach KA;Moghbeli T;Snyder CM
小鼠巨细胞病毒（MCMV）越来越多地被用作研究小鼠中宿主-病原体相互作用的感染模型。已经公布了使用原代鼠胚胎成纤维细胞（MEF）制备MCMV原液和测定病毒滴度的详细方法。为了通过空斑测定法测定MCMV的滴度，这些方法依赖于限制病毒通过培养物上清液传播的高粘度培养基，但通常也太粘而无法移液。此外，MEF必须重复产生，并且在纯度、增殖速率和衰老的发展方面批次与批次之间可能有很大差异。相比之下，M2 - 10B4骨髓基质细胞系（ATCC #CRL-1972）也允许MCMV，据报道可产生高滴度MCMV储备液，并具有快速和持续生长的显著优势。然而，使用这些细胞的详细方法尚未公布。我们修改了现有的方案，使用M2 - 10B4细胞通过空斑试验测量MCMV滴度。我们发现，MCMV斑块可以很容易地解决M2 - 10B4细胞单层。此外，即使在感染当天M2 - 10B4细胞培养物的汇合度小于50%，只要我们还使用降低粘度的覆盖层，噬菌斑也正常形成。总的来说，我们的方案使我们能够使用一致的细胞系来评估病毒滴度，而不是重复产生原代MEF。它还允许我们以比产生融合单层所需的细胞少4倍的细胞开始测定，从而减少测定开始前的准备时间。最后，降低粘度的CMC可以通过移液管处理，并且不需要与培养基预混合，从而增加其保质期和易用性。我们在此描述了我们的结果，沿着了使用M2 - 10 B4细胞系测定MCMV滴度和生长储备液的详细方案。
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. We modified existing protocols to use M2-10B4 cells for measuring MCMV titers by plaque assay. 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. 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.