2.6 NO content measurement
RAW264.7 cells were pretreated with 0/4 μM Tube (MCE, China) for 3 h and stimulated with 1 μg/ml LPS ((Sigma Aldrich, USA) for 0/6/12 h. NO content was measured by mixing 50 μl of medium with Griess reagent [0.1% (w/v) N - (1-naphthyl)ethylenediamine] for 10 min at 37 °C, followed by 50 μl of 1% (w/v) sulfonamide in 5% (v/v) phosphoric acid for 10 min at 37 °C. The absorbance at 540 nm was measured after incubation, and a standard sodium nitrite curve was used to calculate NO concentration.