Synergy2 instrument

All human gut bacterial strains were purchased from the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), with the exception of those we isolated or received from other laboratories, as indicated in the Key Resources Table. The taxonomy of each strain was confirmed by sequencing amplicons generated from their 16S rDNA genes using primers 8F and 1391R. Multiple glycerol stocks of individual strains were frozen to mitigate the need to passage them. Glycerol stocks of Collinsella strains were struck out on Brain Heart Infusion (BHI; Becton and Dickinson) agar plates supplemented with 10% defibrinated horse blood (Colorado Serum Co.) and grown under anaerobic conditions at 37 °C. Single colonies were picked from the plate and inoculated into TYG_s* medium. For experiments involving measurements of growth rates, an aliquot of a stationary phase monoculture was diluted 400-fold into BCM (Table S4A) containing fructoselysine, glucose, and/or lysine at specified concentrations. Growth was quantified in 96-well plates incubated at 37 °C with OD₆₀₀ measurements taken every 15 minutes (BioTek Synergy2 instrument).

Cell lines U87MG were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, L-glutamine (10 mM), penicillin (100 IU/mL), and streptomycin (100 IU/mL). Cells were plated in plated in poly-D-lysine coated 96-well plates at a density of 7,000 cells/well and incubated at 37 °C to allow cells to recover. Culture media was removed after 48 h of incubation, and each well was replenished with DMEM (100 μL) loaded with probenicid (25 μM) and 1x Fluo-4 Direct (from the Fluo-4 NW Calcium Assay Kit, Thermo Fisher), prepared according to the manufacturer’s specifications. The cells were incubated at 37 °C for 30 min to allow the dye to permeate into cells. Extracts or purified compounds (2 μL in DMSO) were added in DMEM (50 μL). Triton-X (50% diluted in DMEM), was added to lyse cell membranes, causing influxes of calcium (positive response), and any unused wells were left blank (negative). After the incubation period, the extract solutions from 96-well round bottom plate were directly added to the plate containing the cells. Relative fluorescence was measured with a BioTek Synergy 2 instrument and Gen5 1.11 software for 1 h, using a G1Ph protocol between the specified filters of 545/40 and 480/40 nm wavelengths for emissions and excitation, respectively.