Griess reagent

The cells were treated with HAGF at various doses (0.5%, 1.0%, 1.5%, and 2.0%), and stimulation was performed with or without LPS. Griess reagents (Promega, Madison, WI, USA) were used to assess the immune-regulatory ability of RAW264.7 cells for NO generation of FAs [31 (link)]. Briefly, 100 μL of the cultured supernatants was combined with 50 μL of Griess reagent A (1% sulfanilamide in 5% phosphoric acid) and 50 μL of Griess reagent B (0.1% N-1-napthylethylenediamine dihydrochloride in water). After incubation, the absorbance of the solution was measured at 540 nm.

Nitric oxide production was verified by measuring its stable end product, nitrite, using a Griess reagent (Promega Corporation, Madison, WI, USA) in the conditioned medium of stimulated and non-stimulated MSCs after 48 h of treatment, according to manufacturer’s protocol. For the assay, 50 μL of the conditioned medium was added to a 96-well plate, followed by 50 μL of sulphanilamide and 50 μL N-1-napthylethylenediamine dihydrochloride (NED). Absorbance at 540 nm was measured by a microplate reader and nitrite concentrations were calculated using a standard nitrite curve.

For fluorescence microscopy, NO was visualized using the NO fluorescent probe 4-Amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM-DA) (Nanjing KeyGen Biotec, Nanjing, China). Leaf fragments were pre-loaded with 5 μM DAF-FM-DA for 30 min in PB (pH 7.4) in darkness at 25 °C. Then, the leaves were washed three times at 25 °C using PB for 5 min each time, and then visualized using a Carl Zeiss laser scanning confocal microscopy 880 (LSMT-PMT, Carl Zeiss, Oberkochen, Germany) with the excitation at 488 nm and emission at 515 nm. To obtain the quantitative data, experiments were performed with the strictly identical confocal settings (e.g., laser power, gain factor, zoom, and emission wave length reception). Graphs represent quantifications from three independent biological experiments. The fluorescence intensity of the individual leaf was determined using Image J software (http://imagej.net/). At least three leaves from different plants were analyzed per experiment. In addition, endogenous NO production was also quantified using the Griess reagent according to the manufacturer’s instruction (Promega, Madison, WI, USA).

U937 or M1 macrophages (2 × 10⁵ cells/mL) were placed in the cell culture plate. To observe the nitric oxide (NO) release during the induction of U937-M1 macrophages, BMSC-Exos (100 μg/mL) or equivalent PBS was added to the culture medium 1 h before the addition of IFN-γ and LPS. NO release was analyzed in the supernatant every 24 h for four consecutive days. The Griess Reagent (Promega, USA) mixed with an equivalent volume of culture supernatant was incubated at room temperature for 10 min. The absorbance was determined by spectrophotometry at 540 nm. The sodium nitrite (NaNO₂) standard curve was used to determine the concentration of NO.