Griess reagent 1

Medium nitrite was measured as an indicator of NO production [40 (link)]. In brief, 50 μl of supernatant was mixed with an equal volume of Griess reagent I, followed by the addition of 50 μl of Griess reagent II (S0021, Beyotime, Shanghai, China) at room temperature. Absorbance was immediately measured at 540 nm. The concentration of each sample was calculated from a standard curve generated using sodium nitrite.

The amount of nitric oxide (NO) produced by BV2 cells is positively related to nitrite release, which is considered an indicator of NO generation. BV2 cells were pretreated with FA (40 μM and 80 μM) for 3 h, followed by 24-h co-exposure to 1 μg/mL of LPS (DH183-1, Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). A volume of 50 μL of supernatant was collected and added to a fresh 96-well plate containing an equivalent volume of Griess reagent I and Griess reagent II (S0021, Beyotime, Shanghai, China). The absorbance was measured at 540 nm using a microwave reader (HBS-1096A, Detie, Nanjing, China).

The production of NO was measured using the Griess method as previously reported with minor modification [39 (link)]. Briefly, the RAW 264.7 cells were pretreated with different concentrations of compounds for 2 h before LPS stimulation. Twenty-four hours after LPS (200 ng/mL) stimulation, 50 μL Griess reagent I and 50 μL Griess reagent II (Beyotime, Shanghai, China) were added into the 50 μL supernatant, respectively. This mixture was incubated for 10 min at room temperature, and the absorbance was measured at 540 nm using a microplate reader (LB 941, Berthold Technologies, Bad Wildbad, Germany). The amount of nitrite in the samples was obtained by a calibration curve using NaNO₂ as the standard.