Protocol for preparing murine fecal microbiota transplant and fecal microbiota metabolites for in vivo and in vitro use

Accumulating evidence demonstrates that microbiota-derived metabolites can modulate host immune responses and influence complex pathologies. Despite their therapeutic potential, the isolation of fecal microbial metabolites and their scalable ex vivo production remain challenging. Here, we present a protocol for preparing murine fecal microbiota transplant (FMT) and fecal microbiota metabolites for in vivo and in vitro use. We describe steps for collecting and preparing donor feces, isolating fecal microbial metabolites, and producing fecal microbial metabolites ex vivo with a broth expansion culture. For complete details on the use and execution of this protocol, please refer to Phelps et al . 1 • Steps for murine fecal bacteria and metabolite isolation • Instructions for murine fecal microbiome transplant (FMT) preparation • Steps for fecal bacteria culture to increase metabolite yield ex vivo Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Accumulating evidence demonstrates that microbiota-derived metabolites can modulate host immune responses and influence complex pathologies. Despite their therapeutic potential, the isolation of fecal microbial metabolites and their scalable ex vivo production remain challenging. Here, we present a protocol for preparing murine fecal microbiota transplant (FMT) and fecal microbiota metabolites for in vivo and in vitro use. We describe steps for collecting and preparing donor feces, isolating fecal microbial metabolites, and producing fecal microbial metabolites ex vivo with a broth expansion culture.