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 ¹³C tracing in bacterial culture with ¹³C-labeled CO₂ 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.