Abstract
In addition to leukocytes, a variety of cells also participate in the innate immune response, including endothelial cells, epithelial cells, and fibroblasts. Thus, the study of these cells is highly relevant in broadening our understanding of mechanisms that modulate innate immunity. With the rise of genetically engineered animals, it is now common to confirm in vitro data acquired using immortalized cell lines with more physiologically relevant primary cells from these animals ex vivo. Indeed, many studies exploring innate immune system function employ mouse embryonic fibroblasts (MEFs). These cells are relatively simple to generate and are a powerful tool to explore regulatory networks, examine biochemical profiling of protein complexes, and investigate novel signaling pathways associated with innate immune system signaling. Here, we provide a robust protocol to isolate, maintain, and store primary MEFs. This protocol is designed for users with minimal experience using mouse models. We have also added precautions and common pitfalls associated with these procedures.
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Davis BK, Wen H, Ting JP (2011) The inflammasome NLRs in immunity, inflammation, and associated diseases. Annu Rev Immunol 29:707–735
Ting JP, Duncan JA, Lei Y (2010) How the noninflammasome NLRs function in the innate immune system. Science 327:286–290
Wen H, Lei Y, Eun SY, Ting JP (2010) Plexin-A4-semaphorin 3A signaling is required for Toll-like receptor- and sepsis-induced cytokine storm. J Exp Med 207:2943–2957
Ishikawa H, Barber GN (2008) STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455:674–678
Seth RB, Sun L, Ea CK, Chen ZJ (2005) Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 122:669–682
Yoneyama M, Kikuchi M, Natsukawa T et al (2004) The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 5:730–737
Kawai T, Takahashi K, Sato S et al (2005) IPS-1, an adaptor triggering RIG-I- and Mda5-vmediated type I interferon induction. Nat Immunol 6:981–988
Meylan E, Curran J, Hofmann K et al (2005) Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437:1167–1172
Allen IC, Moore CB, Schneider M et al (2011) NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-kappaB signaling pathways. Immunity 34:854–865
Acknowledgment
This work is supported by NIH grants R00 DE024173 (YLL), R01 DE026728 (YLL), and Rogel Cancer Center Fund for Discovery (YLL).
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Tan, Y.S., Lei, Y.L. (2019). Generation and Culture of Mouse Embryonic Fibroblasts. In: Allen, I. (eds) Mouse Models of Innate Immunity. Methods in Molecular Biology, vol 1960. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9167-9_7
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DOI: https://doi.org/10.1007/978-1-4939-9167-9_7
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-9167-9
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