Nitrite assay

Raw264.7 cells were seeded at 2 × 10⁴ cells/well in 96-well plates (Costar Corning, Rochester, NY) and incubated in the culture medium overnight. The cells were pretreated with various concentrations of DMF (0, 1.25, 2.5, 5, 10, 20 and 40 μM) for 5 h and then respectively treated with 1 μg/ml LPS for 24, 48 and 72 h. The nitrite accumulation in the supernatant was detected by the Griess reaction kit (#S0021, Beyotime, Shanghai, China). Mixed 50 μl cell supernatant with an equal volume of Griess reagent and incubated at room temperature for 10 min. The absorbance at 530 nm was measured by the Fluoroskan Ascent Fl (ThermoFisher, USA). A series of known concentrations of sodium nitrite was used as standards.