Quantification of Bacterial Signatures

Quantification of flagellin and lipopolysaccharide was previously described using human embryonic kidney (HEK)-Blue-mTLR5 and HEK-Blue-mTLR4 cells, respectively (Invivogen, hkb-mtlr5 and hkb-mtlr4, respectively)^{3 (link),4 (link)}. Fecal material were 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, serially diluted the resulting supernatant, and applied to mammalian cells. Purified E. coli flagellin and LPS (Sigma, L2887) were used for standard curve determination using HEK-Blue-mTLR5 and HEK-Blue-mTLR4 cells, respectively. After 24 h of stimulation, cell culture supernatants were applied to QUANTI-Blue medium (Invivogen, rep-qb1) and measured alkaline phosphatase activity at 620 nm after 30 min