Nitric oxide and proinflammatory cytokine assay

To evaluate the antineuroinflammatory effects of PSE or PSF, BV2 cells (2 × 10⁴ cells/well) were seeded and pretreated with different doses of PSE (10, 20, or 40 μg/ml), three PSF (10 μg/ml) or NAC (20 mM). After incubation for 2 h, the cells were exposed to 1 μg/ml LPS for 24 h. The supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide/0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride/2.5% H₃PO₄). After incubation for 15 min at 37°C, the absorbance was measured at 540 nm using a UV spectrophotometer (Molecular Devices).

The cells were cultured under the same conditions as for the nitric oxide (NO) assay, and the levels of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) in the supernatants were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, San Jose, CA, United States). The absorbance was read at 450 nm using a UV spectrophotometer (Molecular Devices).