V plex system

The ability of the P. distasonis strains to induce the secretion of GLP-1 was measured using the neuroendocrine murine cell line STC-1 (ATCC CRL-3254^™), as previously described [14 (link),35 (link)] Cells were grown at 37 °C under 5% CO₂ in DMEM (Life Technologies, Saint Aubin, France), supplemented with fetal calf serum (10%, Dutscher, Brumath, France), L-glutamine (5 mM) and streptomycin and penicillin (100 µg/mL). For bacterial stimulation, cells were grown in 12-well plates (200,000 cells/well) for 72 h, washed twice with PBS and resuspended in 400 µL of 20 mM Hepes/20 mM Tris pH 7.4 buffer containing 140 mM NaCl, 4.5 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 10 mM glucose. Cells were stimulated (or not) with the bacteria (10:1 bacteria/cell) or with butyrate (10 mM final) as a positive control at 37 °C under 5% CO₂. After 8 h stimulation, the supernatants were collected and centrifuged (10 min at 8000× g). DPP-IV enzyme inhibitor (Ile-Pro-Ile, Sigma-Aldrich Germany) was added (100 µM), and samples were stored at −20 °C. The level of active GLP-1 was measured using the V-Plex system and MESO QuickPlex SQ 120 (MesoScale Diagnostics, Rockville, MD, USA).

The intestinal neuroendocrine murine cell line STC-1 (kindly provided by Dr. Benoit Cudennec Institut Charles Viollette, Lille, France) was grown at 37 °C under 5% CO₂ in DMEM (Life Technologies), supplemented with 10% of fetal calf serum (Dutscher, Brumath, France), 5 mM L-glutamine and 100 µg/mL of streptomycin and penicillin. Cells were seeded in 12 well plates at 200,000 cells/well, grown for 72 h, washed twice with PBS, and resuspended in 400 µL of 20 mM Hepes/20 mM Tris pH 7.4 buffer containing 140 mM NaCl, 4.5 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 10 mM glucose. Cells were subsequently stimulated with the bacteria (10 µL) at a bacteria-to-cell ratio of 10:1 or with butyrate (10 mM final) as a positive control for 8 h at 37 °C under 5% CO₂. The supernatants were then harvested, centrifuged (10 min at 8000× g), and stored at −20 °C. Quantification of active GLP-1 was performed using the V-Plex system and MESO QuickPlex SQ 120 (Meso Scale Diagnostics, Rockville, MD, USA).