Lactobacillus mrs broth

Lactobacillus gasseri (BCRC14619) was cultured in Lactobacillus MRS broth (Merck Millipore, Burlington, MA, USA) at 37 °C, according to the ATCC guidelines. The pelleted bacteria were first dissolved in 1% lysozyme (Sigma-Aldrich, St. Louis, MO, USA) solution to lyse the cells. Ammonium sulfate (Sigma-Aldrich) was then added incrementally to the cytosolic fraction. The precipitated crude extracts were then separated through DEAE-Sepharose ion-exchange chromatography (HiTrap™ DEAE FF; GE Healthcare, Chicago, IL, USA) and further sub-separated through Sephacryl S-300 HR size-exclusion chromatography (Amersham Biosciences, Buckinghamshire, UK). The crude extracts, fractions, and sub-fractions were subsequently co-cultured with mouse bone marrow-derived dendritic cells (BMDC) to screen the active ingredient (detailed information is provided in Additional file 1).

Fresh faeces, small intestinal and caecal contents were collected from P2rx7^−/− mice and resuspended 1:10 (weight: volume) in PBS +0.1% L-cysteine-HCl. The suspensions have been then mixed and tenfold serially diluted. The dilutions were plated on LAMVAB medium^{51 (link)} and incubated at 37 °C under anaerobic conditions (AnaeroGen, Oxoid) in jars (AnaeroJar, Oxoid) for 72 h. Based on the identification of different colony morphotypes, 72 isolates have been picked, re-isolated on LAMVAB medium in order to obtain pure colonies and identified by Sanger sequencing of the 16S rRNA gene (8F: AGA GTT TGA TCC TGG CTC AG; 1391R: GAC GGG CGG TGT GTR CA). The Lactobacillus isolates were grown in Lactobacillus-MRS broth (EMD Millipore, Burlington, MA, USA) at 37 °C under anaerobic conditions. E. coli DH10B was grown aerobically in Luria-Bertani broth (Sigma-Aldrich, Saint Louis, MO, USA) at 37 °C.