Generation and Culture of Mouse Embryonic Fibroblasts.

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.