Griess reagent

Nitric oxide (NO) assay was done in 24-well plates. MDM were treated by adding 1 mL/well of UV-treated supernatant from 1373 or by direct infection with 1373 strain of BVDV (MOI of 1) kept with the cells. Mock-infected supernatant or RPMI 1640 media containing 5% FBS were used as a control. The cells were incubated for 24 h at 37 °C. The cells were then stimulated with lipopolysaccharide (25 µg/mL) (LPS, Sigma, St. Louis, MO, USA) for another 24 h. Then, supernatants were collected and nitric oxide (NO) concentration was determined using Griess reagent (Sigma, St. Louis, MO, USA) using Nitrite (NO₂) measurement as an indicator of NO production according to the protocol of Blond et al., 2000: briefly, 100 µL of culture supernatant was added to 100 µL of Griess reagent, made of a 1/1 mixture of 1% (wt/vol) sulfanilamide and 0.5% (wt/vol) N-(1-naphthyl)ethylenediamine dihydrochloride (Sigma, St. Louis, MO, USA) in 30% acetic acid, in each well of a 96-well plate. Reactions were performed in triplicate at RT for 10 min. Chromophore absorbance was then measured at 550 nm in a microplate reader (Bio-TEK Instruments model EL 311; OSI, Paris, France). Nitrite concentration was evaluated by comparison with the nitrate standard curve (Sigma, St. Louis, MO, USA). The lower limit of detection for nitrite is 250 nM.

The level of NO production was determined in RAW 264.7 cells. Briefly, after cells attachment, they were treated with LPS (Millipore Sigma, Darmstadt, Germany) at 1 µg/mL for 24 h, and then the peptides were added for an additional 24 h [28 (link)]. After cells’ treatment, the medium was collected, and the accumulation of NO metabolite in the cell culture supernatant was measured using Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride; Sigma Aldrich), where 50 µL of the supernatant was mixed with 50 µL of Griess reagent (40 mg/mL) in a 96-well plate. After incubation at room temperature and darkness for 10 min, the absorbance was measured at 540 nm. Cells treated with LPS without samples were used as the positive control of the inflammatory response.

The inhibitory effect of GA on NO production in RAW 264.7 cells was evaluated using the Griess reagent (Sigma–Aldrich Inc.) [23 (link)]. RAW 264.7 cells were seeded into 24-well plates (5 × 10⁴ cells/well) and incubated for 24 h. After incubation, the cells were incubated in the absence and presence of GA (300 and 600 μM) for 2 h. Subsequently, except for the negative control, lipopolysaccharide derived from Escherichia coli O111:B4 (LPS; 1 μg/ml; Sigma–Aldrich Inc.) was added to each well and allowed to react for 22 h. After inducing inflammation using LPS, the supernatant of each well was collected and mixed with the same volume of Griess reagent. The reaction was performed in the dark, and the absorbance was measured at 540 nm using a VersaMax microplate reader (Molecular Devices).

The RAW264.7 cell was seeded at seeding concentration 1 × 10⁴ cells/well. At first, the sample was pretreated with different concentrations [20 (link)]. After 1 h, the cell was stimulated with LPS (1 μg/mL) collected from (Sigma-Aldrich, St. Louis, MO, USA). The cell supernatant was collected after 24 h of incubation. NO (Nitric oxide) production was measured using Griess reagent by colorimetric Griess reaction (100 mL of pre-mixed Griess reagent from Sigma-Aldrich, St. Louis, MO, USA). The equal volume of reagent was added with supernatant and incubated for 10 min to determine NO production. Two multimode microplate readers were used to measure the absorbance at 450 nm for Nitric oxide production assay.

The RAW 264.7 macrophage cells were plated at a density of 2 × 10⁵ cells per well in a 24-well plate. The cells were pre-treated with the indicated concentrations of various extracts for 2 h, and then induced with a 1 μg/mL concentration of LPS for an additional 22 h. Nitrite accumulation in the culture was measured colorimetrically by the Griess reaction using a Griess reagent (Sigma-Aldrich, St. Louis, MO, USA). For the assay, equal volumes of cultured medium and Griess reagent were mixed, and the absorption coefficient was calibrated using a sodium nitrite solution standard (Sigma-Aldrich, St. Louis, MO, USA). The absorbance of each sample after the Griess reaction was determined by an ELISA plate reader at 540 nm.

The content of NO produced from RAW 264.7 cells was measured in the form of NO₂ detectable in the culture supernatant, using the method established by Green et al (16 (link)) with griess reagent (Sigma Chemical Co., St. Louis, MO, USA). RAW 264.7 cells were seeded (5×10⁵ cells/well) in 6-well plates and washed twice with 1× PBS (Thermo Scientific HyClone, Logan, UT, USA) after culturing in the CO₂ incubator at 37 °C for 24 hours. LPS (1 μL/mL) (Sigma Chemical Co., St. Louis, MO, USA) was treated with a sample solution of each concentration 2 hours after removal, cultured for 24 hours to obtain the supernatant 100 μL, reacted for 10 minutes in a 96-well plate by adding the same amount of griess reagent, and the optical density was measured at 540 nm. The level of NO inhibition activation was expressed as optical density reduction ratio of specimen added group and non-specimen adding group.

The effect of BVDV-infected MDM supernatant on nitic oxide activity of neutrophils was examined using the Griess reagent. Briefly, freshly collected neutrophils were treated with 3 BVDV strain-specific MDM supernatants as well as mock-treated control and incubated for 6 h at 37 °C. Cell samples from each treatment were then stimulated with LPS (25 µg/mL) (Sigma, St. Louis, MO, USA) for 1 h. Then, supernatants were collected, and nitric oxide (NO) concentration was determined using Griess reagent (Sigma, St. Louis, MO, USA) using Nitrite (NO2) measurement as an indicator of NO production according to [21 (link)]. Briefly, in each well of a 96-well plate, a 100 µL of culture supernatant was added to 100 µL of Griess reagent, which was made of a 1:1 mixture of 1% sulfanilamide and 0.5% N-(1-naphthyl) ethylenediamine dihydrochloride (Sigma, St. Louis, MO, USA) in 30% acetic acid. Reactions were performed in triplicate at RT for 10 min. Absorbance was then measured at 540 nm in a microplate reader (Biotek, ELx808, Winooski, VT, USA). Nitrite concentration was evaluated by plotting the average OD values of each sample (in triplicates) against sodium nitrite standard curve (Sigma, St. Louis, MO, USA) with a detection range of 1.95–200 μmol/mL.