TNF-α ELISA assay

As described previously (23), differentiated Caco-2 cells in 24-well plates were incubated with QHCY (10 and 50 µg/ml) for 1 h before stimulation with LPS (1 µg/ml) for 24 h at 37°C. Subsequently, the supernatants were collected by centrifuging the cell culture medium at 3,000 × g for 10 min at room temperature. Using a human TNF-α ELISA kit, the production of TNF-α from Caco-2 cells was measured, according to the manufacturer's instructions. Absorbance was read at 450 nm using a fluorescence plate reader. All samples were assessed in triplicate.