4.5. ELISA Assay

The cells were seeded in 12-well plates at 2.0 × 105 cells/mL and cultured for 3 days at 37 °C. On the day of the experiment, 1 µg/mL of LPS or CX at 3.8 µg/mL was added to the culture medium with or without various concentrations of CLE (0.1–1000 µg/mL) for 24 h at 37 °C. The treated cells were then taken for the next assay and centrifuged at 2000× g for 10 min at 4 °C. The supernatant was collected for determining the IL-6, IL-1β, TNF-α, NO, and PGE2 concentrations using commercial kits, according to the manufacturer’s instructions (BioLegend, San Diego, CA, USA). The levels of inflammatory cytokines were measured at a wavelength of 540 nm using a SynergyTM HT microplate reader (Biotek, Winooski, VT, USA).