Griess reagent system kit

Nardochinoid B (NAB) (Figure 1) (HPLC purity >98%) was provided by the Institute of Traditional Chinese Medicine and Natural Products, Jinan University (Guangzhou, China). Dimethyl sulfoxide (DMSO) (Sigma, Cat. No. D2625, St. Louis, MO, USA) was used to dissolve the NAB powder to give a stock solution of 30 mM concentration. Lipopolysaccharide (LPS), dexamethasone (DEX), and sulforaphane (SFN) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies to iNOS [57 (link)], COX-2 [58 (link)], heme oxygenase (HO)-1 [58 (link)], Nrf2 [18 (link)], p-p65 [18 (link)], p-p38 [18 (link)], and phospho-extracellular regulated protein kinases (p-ERK) [18 (link)] were obtained from Cell Signaling Technology (Boston, MA, USA). Antibodies to β-actin and laminin B1 [18 (link)] were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to α-tubulin [18 (link)] was from Sigma Chemical Co. (St. Louis, MO, USA). The secondary antibodies for Western blot were from Li-COR Biotechnology (Lincoln, NE, USA). ELISA kits for IL-1β, IL-6, and TNF-α was from eBioscience (eBioscience, Inc., San Diego, CA, USA). ELISA kit for PGE₂ was from Cayman Chemical (Cayman Chemical, Ann Arbor, MI, United States). The nitric oxide (NO) production level was measured by a Griess Reagent System kit, which was obtained from Promega Corporation (Madison, WI, USA).

The anti-inflammatory activity of the extracts (400 to 6.25 μg/mL) was evaluated based on nitric oxide (NO) production in a RAW 264.7 murine macrophage cell line (ECACC 91062702) due to lipopolysaccharide (LPS, 1 mg/mL in DMEM; Sigma-Aldrich, Saint Louis, MO, USA) stimulation. For that purpose, the Griess Reagent System kit (Promega, Madison, WI, USA) was used as described in previously published protocols [32 (link)]. Dexamethasone (50 mM) (Sigma-Aldrich, Saint Louis, MO, USA) and samples without the addition of LPS were used as positive and negative controls, respectively. The NO generated was monitored at 540 nm (ELX800 Biotek microplate reader; Bio-Tek Instruments Inc., Winooski, VT, USA). The extract concentrations that trigger 50% of NO production inhibition were expressed as EC₅₀ values (μg/mL).

Apamin (purity 98.3%) was obtained from CalBiochem (San Diego, CA, USA), while melittin (purity ≥ 85%, HPLC), PLA2 from bee venom (activity: 1775 units mg⁻¹ solid), cytochrome c from the equine heart (purity ≥ 95%), lipopolysaccharide (LPS), ellipticine, dexamethasone (DM), sulforhodamine B, trypan blue, trichloroacetic acid (TCA), and tris were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Formic acid was from Panreac (Barcelona, Spain). Standards for metal analysis were purchased from PanReac (Barcelona, Spain). The Griess Reagent System Kit was purchased from Promega (Madison, WI, USA). Dulbecco’s Modified Eagle’s medium (DMEM), Hank’s balanced salt solution (HBSS), Foetal bovine serum (FBS), L-glutamine, trypsin-EDTA, penicillin/streptomycin solution (100 U/mL and 100 mg/mL, respectively) were purchased from TermoFisher Scientific (Waltham, MA, USA). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Sunnyvale, TX, USA).

The procedure described by Sobral et al. [22 (link)] to measure the inhibition of nitric oxide (NO) produced by LPS-stimulated RAW 264.7 macrophages was followed to evaluate the anti-inflammatory potential of fish oils. The NO quantification was performed using a Griess Reagent System kit (Promega, Madison, WI, USA). Fish oils were dissolved in DMSO:H₂O (1:1 v/v) at 8 mg/mL and successively diluted to the concentrations (6.25–400 µg/mL) to be tested. Dexamethasone at 50 µM was used as a positive control and oil samples without LPS were employed as negative controls. Results were expressed as the oil concentration (μg/mL) that caused 50% of NO production inhibition (IC₅₀ value).

The concentration of nitrite, a stable oxidized product of NO, in the cell culture medium was determined using a Griess reagent system kit (Promega). Cells were seeded in 96-well plates (2 × 10⁴ cell/well) and treated with the positive control or different concentrations of white mulberry fruits crude extract and fractions for 2 h, followed by LPS for 24 h. Then, 50 μL samples of the supernatant from the treated culture medium was mixed with 50 µL 1% sulfanilamide in 5% phosphoric acid and incubated at room temperature for 10 min, protected from light. Then, 50 µL 0.1% N-1-napthylethylenediamine dihydrochloride in water was added, followed by incubation at room temperature for 10 min, protected from light. The absorbance was measured at a wavelength of 540 nm using a microplate spectrophotometer. The NO level of each experimental sample was calculated using a NaNO₂ (0–100 µM) standard curve.

MDSCs were lysed with an appropriate amount of RIPA buffer for 30 min, and the lysate supernatant was collected after centrifugation. A QuantiChrom arginase assay kit (BioAssay systems, Hayward, CA) was used to determine the Arg1 activity in the lysate supernatant.
The content of NO was measured by the Griess reagent system kit (Promega, Madison, WI) according to the manufacturer's instructions.

Tween^® 20, cholesterol, cetyl alcohol, chloroform, sulforhodamine B, lipopolysaccharide, trypan blue, dexamethasone, trichloroacetic acid, tris (hydroxymethyl)aminomethane buffer, cytochrome C from equine heart (purity ≥ 95%), melittin (purity ≥ 85%, HPLC grade) and phospholipase A2 (activity: 1775 units/mg solid) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Apamin (purity 98.3%) was purchased from CalBiochem (San Diego, CA, USA). Fetal bovine serum, penicillin, streptomycin, trypsin, L-glutamine, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco Invitrogen Life Technologies (Carlsbad, CA, USA). Formic acid (HPLC grade) and acetonitrile (HPLC grade) were obtained from Fisher Scientific (Loughborough, UK). The Griess reagent system kit was bought from Promega (Madison, WI, USA). Ultrapure water was obtained from adequate purification systems (Ellix Essential Millipore^®, Darmstadt, Germany, and TGI Pure Water Systems, Brea, CA, USA).