4.10. Measurement of NO, TNF-α, IL-6 Cytokine Production

After being cultured for 24 h in 96-well plate (1 × 10⁶ cells/well) at 37 °C in a humidified incubator, DMEM medium in the presence or absence of PSPs (50, 500, 2000 μg/mL) or LPS (10 μg/mL) were added to RAW264.7 cells culture wells followed by incubation for another 24 or 48 h. Then, the culture medium containing NO and cytokines secreted from active macrophage RAW264.7 cells were collected for further analysis. The levels of NO and cytokines were measured using nitric oxide assay kit, TNF-α, and IL-6 ELISA kits, respectively. LPS was used as a positive control. The DMEM medium with no PSP was used as blank control. All pre-experiments were done to confirm that the dilution rate was appropriate for kit tests.