Nitric Oxide Quantification in BV-2 Cells

BV-2 microglial cells were purchased from Peking Union Medical College Cell Bank (Beijing, China) and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, penicillin G (100 U mL⁻¹), and streptomycin (100 μg mL⁻¹) at 37 °C in a humidified incubator with 5% CO₂. The NO concentration was detected by the Griess reagent.^{33 (link)} Quercetin was used as a positive control. BV-2 cells were seeded at the density of 1.5 × 10⁵ cells per mL in 96-well culture plate and treated with each compound and LPS (1.0 μg mL⁻¹) for 24 h. After that, 50 μL of cell-free supernatant was allowed to react with an equal volume of Griess reagent (Beyotime Institute of Biotechnology, Shanghai, China) for 15 min at room temperature in the dark. Then, the absorbance was measured at 540 nm using a microplate reader. The cell viability of the cultured cells was detected by MTT-based colorimetric method.