4.6. Nitric Oxide Assay

The cells were plated in a 12-well plate at 2.0 × 105 cells/mL and cultured for 3 days at 37 °C. On the day of the experiment, culture media with or without various concentrations of CLE (0.1–1000 µg/mL) or CX at 3.8 µg/mL, with or without 1 µg/mL of LPS, were added for 24 h at 37 °C. The treated cells were then taken for the next assay and centrifuged at 10,000× g for 20 min at 4 °C. The supernatant was subsequently collected for measurement of the nitric oxide concentration. The nitric oxide level was measured at a wavelength of 540 nm using a SynergyTM HT microplate reader (Biotek, Winooski, VT, USA).