Quantifying Fecal Flagellin and LPS Levels

Levels of fecal bioactive flagellin and lipopolysaccharide (LPS) were quantified as previously described [13 (link)] using human embryonic kidney (HEK)-Blue-mTLR5 and HEK-Blue-mTLR4 cells, respectively (Invivogen, San Diego, CA, USA) [13 (link)]. MBRA samples (whole suspension without centrifugation) were serially diluted and applied to mammalian cells. For fecal samples, fecal material was resuspended in PBS to a final concentration of 100 mg/mL and homogenized for 10 s using a Mini-Beadbeater-24 without the addition of beads to avoid bacteria disruption. Samples were then centrifuged at 8000 × g for 2 min and the resulting supernatant was serially diluted and applied to mammalian cells. Purified E. coli flagellin and LPS (Sigma-Aldrich) were used for standard curve determination using HEK-Blue-mTLR5 and HEK-Blue-mTLR4 cells, respectively. After overnight incubation at 37 °C, the cell culture supernatant was applied to QUANTI-Blue medium (Invivogen), and the alkaline phosphatase activity was measured at 620 nm after 30 min.