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
Hkb mtlr5
Manufactured by InvivoGen
Hkb-mtlr5 is a laboratory equipment product offered by InvivoGen. It serves as a core functional component without further interpretation of its intended use.
Automatically generated - may contain errors
3 protocols using hkb mtlr5
1
Quantification of Bacterial Signatures
2
SFB Culture Flagellin Bioactivity Assay
3
SFB Culture Flagellin Bioactivity Assay
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Equal numbers of IO-only and filament enriched SFB fractions, based on 16S rDNA qPCR analysis, from 3-day old in vitro-grown SFB cultures were sonicated for 10 min (Bioblock, 88154) and 1/5 dilutions of lysate were added to control and mTLR5-expressing HEK cells in HEK-Blue detection medium (Invivogen, hkb-null1v, hkb-mtlr5, hb-det2) seeded in 96-well plates for 16 hours before alkaline phosphatase activity was measured at 620 nm using an ELISA plate reader (Labsystem Multiscan RC). Purified Salmonella Typhimurium flagellin (Invivogen, tlrl-stfla) served as the positive control.
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