Sodium nitrite solution

Nitrite incubations were initiated after 5 minutes initial incubation of the RBC mixture in the tonometer, to equilibrate the samples to temperature and gas conditions. Before addition of nitrite, gas was collected for background NO values. Next, 100 µl of a sodium nitrite solution (Sigma-Aldrich) was added to the blood sample by pipetting directly into the cuvette using an elongated pipet tip. This allowed addition without changing the gas-state in the tonometer. At set time points (generally after 0, 10, 20, 40, 60, 80 and 100 minutes) gas outflow samples were taken to measure NO release using mylar balloons, with gas collection for 2 minutes. The NO in the mylar balloons was determined similar the to analysis of NO in exhaled air by the Sievers nitric oxide analyzer (NOA 280, GE Water & Process Technologies, Trevose, PA, USA) (See Figure 1B). All NO peaks were recorded by the NOAnalysis™ software. This allowed peak quantification (see example Figure 1C). Finally, NO values collected are used to calculate the NO release rate (see example Figure 1D) and to calculate the sum of total mole NO (see example Figure 1E) released per mole hemoglobin during the run.

RAW264 cells were prestimulated with 50 ng/mL receptor activator of nuclear factor κβ ligand (RANKL) (PeproTech, Rocky Hill, NJ, USA) for 24 h. Mouse bone marrow-derived osteoclast precursor cells were prestimulated with 50 ng/mL RANKL and 25 ng/mL macrophage colony-stimulating factor (M-CSF) (Sigma-Aldrich) for 24 h. Then, the cells were stimulated with 10 ng/mL, 100 ng/mL, 1 μg/mL, and 10 μg/mL Mfa1 and FimA fimbriae or 50 ng/mL RANKL every 48 h. After 96 h, the cells were washed with phosphate-buffered saline and fixed in 3.3% formaldehyde, a citric acid solution, and acetone for 5 min. The staining solution was prepared by mixing a Fastgarnet GBc BASE solution, sodium nitrite solution, Naphthol AS-BIPb, acetic acid solution, tartaric acid solution (all purchased from Sigma-Aldrich), and distilled water. After fixation, cells were washed with distilled water and stained in the solution for 30 min. After the cells were washed with distilled water and dried, TRAP-positive multinucleated cells containing three or more nuclei were counted under an optical microscope (BZ-X700, KEYENCE, Osaka, Japan).

Soy protein isolate (90 % protein content), was purchased from Kemin Industries (Herentals, Belgium). Ethanol 96 % was purchased from Titolchimica (Italy). Apple pectin, sodium carboxymethyl cellulose, HPLC purity methanol, 2,2-diphenyl1-picrylhydrazyl (DPPH), Folin–Ciocâlteu reagent, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), gallic acid, sodium nitrite solution, glacial acetic acid, potassium chloride solution, sodium acetate solution, sodium hydroxide, aluminum chloride, sodium carbonate, hydrochloric acid, sodium bicarbonate were acquired from Sigma-Aldrich (Steinheim, Germany).

Diphenyleneiodonium (DPI), 2′,7′-dichlorofluorescin diacetate (DCFA), lipopolysaccharide (LPS), sodium nitrite solution, Griess reagent, Dulbecco’s Modified Eagle Medium (DMEM)/low Glucose, phenol red-free DMEM/low Glucose, penicillin/streptomycin mixture, DMSO, and fetal bovine serum (FBS) were purchased from Sigma (St. Louis, MO, USA). Glucose was purchased from Acros Organics (Fair Lawn, NJ, USA), and sodium bicarbonate was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ, USA). Caco-2 cells and RAW 264.7 macrophages (cell line TIB-71™) were acquired from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and phosphate-buffered saline (PBS) for cell culture were purchased from Gibco-BRL (Grand Island, NY, USA). Lipopolysaccharide (LPS), thiazolyl blue tetrazolium bromide (MTT), Griess reagent, sodium nitrite solution, the 19 selected volatile compound standards (α-pinene, camphene, β-pinene, sabinene, 3-carene, β-myrcene, α-phellandrene, α-terpinene, limonene, p-cymene, terpinolene, 2-nonanone, acetic acid, citronellal, bornyl acetate, thujopsene, β-chamigrene, (-)-β-bisabolene, and nerolidol) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). In addition, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) was obtained from Thermo Fisher Scientific (Carlsbad, CA, USA). Antibodies against iNOS, p65, phospho-p65 (p-p65), and β-actin were obtained from Santa-Cruz Biotechnology (Dallas, TX, USA). Materials for western blot were obtained from Bio-Rad (Hercules, CA, USA), and all other reagents and chemicals used were of analytical grade.