Determination of TNF-α and IL-6

RAW 264.7 cells were cultured in 96-well plates at a density of 2×10⁵ cells/well. The cells were treated with LPS (1 µg/ml) with or without tiliroside (12.5, 25, 50 and 100 µM) and then incubated for 6 h. Cell culture supernatant (100 µl) was removed to determine the levels of TNF-α or IL-6 by using respective mouse TNF-α (SEM024) and mouse IL-6 (SEM008) enzyme-linked immunosorbent assay (ELISA) kits (Yantai Science & Biotechnology, Co., Ltd.), as previously described (19).