Abstract
Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Because of their ubiquitous distribution as tissue cells, these cells are poised to respond to factors released by newly activated innate immune cells, thus becoming a useful tool to study inflammation and immunity. Here, we describe procedures for mouse embryonic fibroblast cell isolation, primary culture, and stimulation. Specifically, we have optimized a step of serum starvation prior to stimulation. This step is necessary to maintain the quiescent status of these cells before they are exposed to pro-inflammatory stimuli for optimal responses. As shown in our previous studies, these mouse fibroblasts do not express Tnf, Csf2 or Il2 mRNAs at levels readily detectable by routine northern blotting techniques (Lai WS et al., 2006).
Keywords: Mouse embryonic fibroblast, Primary cell culture, Tumor necrosis factor, Serum starvation
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Time courses of induction of TNF-responsive genes in mouse fibroblasts. Expression of Fos and Cxcl1 mRNAs in serum-deprived MEFs from wild-type (WT) and tristetraprolin (TTP)-deficient (KO) mice was examined at various time points after addition of TNF at 10 ng/ml, using real-time RT-PCR. Results shown are means ± SEM of five independent experiments, and data are expressed as fold changes relative to WT at time 0; this value was set as one after normalizing to an internal control, Actb mRNA. Statistical differences between the WT and KO means, determined by paired Student t tests, are indicated (*P < 0.05, WT vs. KO). Modified from Qiu et al. (2015) with permission.
Recipes
Acknowledgments
This protocol was adapted from previously published studies, Lai et al. (2006) and Horner et al. (2009), and was used in Qiu et al. (2015). We thank Dee Wenzel for husbandry support, Christopher McGee for assistance with live animal imaging, and Drs. Melissa Wells and Diana Cruz-Topete for comments on the protocol. This research was supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health.
References
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