Generation and culture of mouse embryonic fibroblasts.

The innate immune system is evolutionarily conserved and shared by a wide spectrum of cells, including epithelial cells, endothelial cells, fibroblasts, macrophages, dendritic cells, and lymphocytes. The extensive utilization of genetically manipulated animals in innate immunity studies has become the standard approach to confirm functional data acquired in cell lines. The easy generation and physiological relevance of mouse embryonic fibroblasts (MEFs) have made them a powerful tool in discovering novel signaling pathways, investigating regulatory networks, and exploring biochemical profiling of protein complexes involved in innate immune responses. Due to their extensive use, this chapter aims to provide a protocol for generating, maintaining, and storing primary MEFs for those who have minimal experience in animal models. Precautions and notes are integrated into the description of each step of the protocol for the benefits of minimizing unnecessary cross-referencing.