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

Abstract

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.

Keywords: metabolism; metabolomics; microbiology; model organisms; molecular/chemical probes.

Graphical abstract

Figure 1

Broth media and supported phyla We use the indicated broth media for fecal bacterial culture to produce cultured microbiota metabolites. The bacterial phyla supported by and the unique properties of each broth are indicated. This figure is adapted from our previous protocol, Phelps and Shapira et al., 2023.

Figure 2

Fecal microbiota transplant (FMT) solution preparation (A) An equal number of pellets from each donor mouse is pooled together in a 50 mL conical tube. (B) Feces are covered with sterile PBS. (C) Feces are hydrated after 1 h incubation in sterile PBS. (D) Feces after mechanical homogenization. (E) Feces after centrifugation, showing upper layer primarily containing bacteria and soluble factors and lower layer primarily containing large debris. (F) Fecal material remaining after filtration step. (G) FMT solution after filtration is opaque brown with no solid debris.

Figure 3

Syringe filter handling Sterile syringe filter should only be handed by the edges between filtration steps as shown.