Protocol for determining gut microbiota metabolites as substrates in mouse metabolism

Here, we present a protocol for studying how microbiota-derived substrates fuel host mouse metabolism using stable isotope tracing. We describe steps for integrating 13 C tracing in bacterial culture with 13 C-labeled CO 2 tracing in free-moving mice. We then detail procedures for determining the metabolic flux and fate of gut microbiota-derived substrates in the host through comparison between germ-free mice and monocolonized or specific pathogen-free mice. This protocol provides a framework for determining metabolic interactions between the gut microbiota and host. For complete details on the use and execution of this protocol, please refer to Fang et al. 1 • Steps for setting up a stable isotope gas analyzer in free-moving mice • Instructions for substrate administration and 13 CO 2 measurement in expired breath • Guidance on data analysis integrating 13 C tracing in culture and mice Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for studying how microbiota-derived substrates fuel host mouse metabolism using stable isotope tracing. We describe steps for integrating 13 C tracing in bacterial culture with 13 C-labeled CO 2 tracing in free-moving mice. We then detail procedures for determining the metabolic flux and fate of gut microbiota-derived substrates in the host through comparison between germ-free mice and monocolonized or specific pathogen-free mice. This protocol provides a framework for determining metabolic interactions between the gut microbiota and host.