Cell Culture for In Vitro Anti-Inflammatory Assay

Mouse macrophage cells (RAW 264.7, ATCC, Manassas, VA) were used to test the selected metabolites' in vitro anti-inflammatory effects. The passage number of the macrophage cells was between 3 and 5. Approximately 1.0 × 10⁵ macrophage cells were seeded in six well plates (Corning Life Sciences, Tewksbury, MA) and cultured with 2 mL of Dulbecco's Modified Eagle Medium (DMEM, Corning Life Sciences, Tewksbury, MA) containing 10% fetal bovine serum (FBS, R&D systems, Flowery Branch, GA). After the cell reached ∼75% of confluence (judged by the percentage of the plate bottom area covered with cells), the culture medium was replaced by a new DMEM medium (with 10% FBS) with or without tuberonic acid and incubated for another 12 h. 6-Shogaol containing DMEM (with 10% FBS) was used as the positive control. After the treatment, the culture medium was removed, and cells were activated by adding diluted lipopolysaccharide (LPS) in the medium (200 ng/mL of LPS, without FBS) and incubated for another 4 h at 37°C.