Lps derived from e coli o55 b5

RAW 264.7 cells (Korean Cell Line Bank, South Korea) were seeded into 24-well culture plates (Corning, USA) at 2.2 × 10⁵ cells/well and incubated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and 1% (vol/vol) antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37°C in 5% CO₂ incubator for 24 h. Confluent macrophage cells were treated with the cell lysate of 100 isolated gut bacteria for 4 h, and then 100 ng/mL LPS-derived from E. coli O55:B5 (Sigma-Aldrich, USA) was added to each well, and the culture plates were incubated at 37°C for 20 h. All supernatant samples were collected (2,000 rpm, 5 min, 4°C).

Sodium butyrate (Sigma-Aldrich, St. Louis, MO) was dissolved in DMEM/F12 to make the butyrate working solution. Cells were treated with butyrate throughout the differentiation period (day 0 to 7). On day 7 post-differentiation, fresh medium was added and cells were challenged with LPS-derived from E. coli O55:B5 (Sigma-Aldrich, St. Louis, MO) at 10 μg/ml. Preliminary experiments were also conducted to determine the optimal butyrate concentration and duration of exposure to LPS to determine effect on cell viability. Analysis of cell viability was performed in 96-well cell culture plates (Corning Inc., Corning, NY) with the use of CellQuanti-Blue^TM cell viability assay kit (BioAssay Systems, Hayward, CA), according to the manufacturer’s instruction. The results of pilot experiments indicated that optimal concentrations of butyrate ranged from 0.1 mM to 1 mM because there was no significant adverse effect on cell viability at these concentrations (S1 Fig). Stimulation with LPS at 10 μg/ml LPS did not cause any cytotoxic effects up to 24 h of culture. There were 4 to 6 replicates per experiment.