4.3. In Vitro Anti-Inflammation Effects on LPS-Induced RAW 264.7 Cells

The murine macrophage (RAW 264.7) cell line was cultured in DMEM high glucose medium supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in an atmosphere of 5% CO₂. The RAW 264.7 cells were seeded in 96-well plates with 1 × 10⁴ /well overnight and then treated with LPS (1.0 μg/mL) for 30 min, followed by incubation with either NB or SB (50 mg/mL) for an additional 4 h or 24 h.

The NO production in the cell-culture supernatant was determined by Griess reagent. Briefly, the culture media was collected and centrifuged at 10,000 rpm, 4 °C for 10 min. Then, 50 µL of Griess reagent was added into 50 µL of culture supernatant, following treatment according to the manufacturer’s instructions. Absorbance of each well was measured at 540 nm using ELISA micro-plate reader. Drew the standard curve with NO content as abscissa, absorbance OD as ordinate. According to the absorbance of the samples’ OD, the concentration of samples can be checked on the standard curve (μM). Similarly, the concentrations of pro-inflammatory cytokines including TNF-α and IL-6 in the cell-culture supernatant were determined by ELISA assay according to the manufacturer’s instructions. The absorbance of each well was determined at a maximum absorption wavelength of 450 nm and reference wavelength of 570 nm with dual wavelength detection by using a micro-plate reader. The sample concentration was calculated according to the standard curve.