E coli lps

Briefly, 24-well microplates were inoculated with 5 × 10⁴ HT-29 cells/mL. The plates were then incubated at 37 °C under a 5% CO₂ atmosphere until reaching confluence. HT-29 cells were then washed two times with PBS and stimulated with E. coli LPS (Sigma Aldrich, Saint Quentin Fallavier, France) and P. distasonis at a final concentration of 10⁷ CFU/mL. To define E. coli LPS concentration to use, assays ranging from 1 ng/mL to 100 ng/mL were performed. The final concentration selected was 1 ng/mL, which induced the same response as other concentrations (data not shown). After a 4 h incubation with LPS and bacteria, the supernatants were collected and stored at −20 °C until cytokine (IL-8) measurement, performed with an IL-8 human ELISA kit (Thermofisher Scientific, Illkirch, France).
For PBMC stimulation, 24-well microplates were inoculated with 1 × 10⁶ of the extracted cells/mL. PBMCs were stimulated with 100 ng/mL of E. coli LPS (Sigma-Aldrich, Saint Quentin Fallavier, France) and P. distasonis at a final concentration of 10⁷ CFU/mL. After 18 h incubation with LPS and bacteria, the supernatants were collected and stored at −20 °C until cytokines’ (IFN-γ, IL-1β, IL-1, IL-12p70, TNF-α, IL-1RA, and IL-10) measurement with a Milliplex Luminex^® 200™ System (Merck Millipore, Molsheim, France) using the Luminex™ Xmap (Multi-Analyte Profiling) technology.

C57BL/6 mice and Sprague-Dawley (SD) rats were housed under 12 h light and 12 h dark for one week with food and water available ad libitum. The animal experimental protocols were approved by the Animal Care and Use Committee of Zhejiang University (Ethic Committee approval number: ZJU20160396).
Mice were injected with 100 μL sterile saline solution containing 5 mg/kg of E. coli LPS (Sigma–Aldrich, St. Louis, MO, USA) intraperitoneally. After one night, the mice were given 50 μg Bodipy-C16:0 (Life Technologies, CA, USA) or 200 μL olive oil, orally. The whole small intestines of Bodipy-C16:0-given mice were collected to estimate FA absorption by fluorescence intensity 3 h later. The blood was collected and the serum was separated by centrifugation at 4 °C, 2000× g, for 15 min. Tissue and contents of the intestines of the mice given olive oil were collected 3 h later for further experiments.
Rats were injected with 1 mL sterile saline solution containing 5 mg/kg of E. coli LPS (Sigma–Aldrich, St. Louis, MO, USA) intraperitoneally. After a night, the rats were given 2 mL olive oil. The blood of the rats was collected 3 h later, and the serum was separated by centrifugation at 4 °C, 2000× g, for 15 min.