L glutamine

Human SH-SY5Y cells (LGC Standards S.r.l., Sesto San Giovanni, Italy) were cultured as monolayers in polystyrene dishes in Dulbecco's modified Eagle's medium (DMEM) containing 15% heat-inactivated fetal bovine serum, 1% L-glutamine (200 mM), 1% sodium pyruvate (100 mM), 100 IU/ml penicillin, and 100 μg/ml streptomycin. All of the above reagents were purchased from Invitrogen (Milan, Italy). Cells were grown in a humidified incubator at 37°C in a 5% CO₂ atmosphere, and the medium was changed every 2 days. Each experiment was performed using cells (passages 15–30) plated on multiwell plates. After 24 h and 48 h of cell seeding, cells were incubated with A1254 (stock solution; 1 mg/ml) in DMEM without serum.

The human alveolar epithelial cell line A549 and the murine macrophage RAW 264.7 were purchased from the American Type Culture Collection (ATCC). The cells were maintained, respectively, in DMEM/F12 and RPMI-1640 culture medium containing 2 mM L-Glutamine (LGC Biotechnology, SP, Brazil), and supplemented with 10% of fetal bovine serum (FBS) (Cultilab, SP, Brazil). Cultures were kept at 37°C within a humidified 5% CO₂ atmosphere. The A549 cells were seeded for 24 hours at 37°C prior to stimulation in 6-well plates (5 x 10⁵ cells for well) for the PCR assays and 24-well plates (7 x 10⁴ cells for well) for the other assays. In the current study, we also used primary nasal epithelial cells obtained from the nasal polyps of patients submitted to polypectomy for nasal clearing (further details see [12 (link)]). The procedures described for the use of nasal polyps were approved by the Pedro Ernesto University Hospital, the State University of Rio de Janeiro, and the Oswaldo Cruz Foundation (FIOCRUZ) Ethical Committee located in Rio de Janeiro, RJ, Brazil. All participants provided their written consent.

Sodium nitrate (NaNO₃), sulfuric acid (H₂SO₄ – 95.0–98.0%), potassium permanganate (KMnO₄), hydrogen peroxide (H₂O₂ – 30 wt%), hydrochloric acid (HCl – 36 wt%), sodium iodide, potassium iodide, iodine, acetic acid (99.5%), sulfanilamide, and MTT assay were purchased from Thermo Scientific, Fisher Scientific, Acros, Nacalai Tesque and Alfa Aesar, respectively. Graphite flakes, N-(3-dimethylaminopropyl-N′-ethylcarbodiimide) hydrochloride (EDC), N-hydroxysuccinimide (NHS), phosphate buffered saline (PBS), potassium bromide (KBr), sodium nitrite (NaNO₂ – 97%), potassium iodide, vanadium chloride, TEM copper grid, cysteamine, S-nitroso-N-acetyl-dl-penicillamine (SNAP), diethyldithiocarbamic acid sodium salt (DETC), iron (II) sulfate heptahydrate (FeSO₄·7H₂O, ≥99%), dichloromethane (CH₂Cl₂), mercuric chloride (HgCl₂), 1 M hydrochloric acid (HCl) and the LDH assay kit (coulorimetric, MAK066) were purchased from Sigma Merck. ATCC medium containing 4 mM l-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate was purchased from LGC Limited, UK. cGMP ELISA kits were purchased from Enzo Life Science, UK. BrdU cell proliferation assay kits were purchased from Cell Signalling Technology, UK. DAF-FM DA was purchased from Abcam, UK. All materials were used as received without any further purification unless stated otherwise.