Commensal-derived acetylcholine enhances mucosal immune education

Bacterial growth and generation of in vitro bacterial metabolite libraries Based on phylogenetic diversity, 100 bacterial strains were picked from the HMP bacterial culture collection and our laboratory’s internal human gut bacterial culture collections (Supplementary Table 1 ). Anaerobic bacteria were cultured in a vinyl anaerobic chamber (Coy Laboratory Products), containing 4% H 2 , 10% CO 2 and 86% N 2 . Aerobic bacteria were cultured in an aerobic microbiological incubator. All bacteria were cultured in triplicate at 37 °C for 48 h. To extract bacterial metabolites with an acetonitrile-methanol-water (2:2:1) mixture (ACN-MeOH-water), the bacterial cultures were dried overnight at room temperature under vacuum (GeneVac). Then, the dried matter was resuspended in ACN-MeOH-water with intermittent sonication. The suspensions were centrifuged at 4,000 g for 10 min, and the supernatant was dried overnight at room temperature under a vacuum (GeneVac). The dried extracts were dissolved in sample buffer (PBS containing 10% DMSO, v/v). To extract bacterial metabolites with butanol or ethyl acetate, the bacterial cultures were mixed with two volumes of butanol or ethyl acetate and shaken vigorously for 10 min in a horizontal shaker at room temperature. The organic phase was collected and dried overnight at room temperature under a vacuum (GeneVac), and the dried extracts were dissolved in sample buffer. …