Antioxidant nac

Lutein, purchased from Cayman Chemical Co. (Catalog No. 10010811, Ann Arbor, MI, USA), was dissolved in dimethyl sulfoxide (DMSO) according to the product information (Cayman Chemical Co, Ann Arbor, MI, USA), aliquoted, and stored at −80 °C. Final concentrations of 5, 10, or 20 μM were applied to the cells. NADPH oxidase 1 inhibitor ML171 and etoposide were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DMSO. Antioxidant NAC (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in distilled water. For each experiment, cells incubated with DMSO alone (less than 0.3%) served as the control.

The MH-S murine AM cell line (CRL-2019) was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin-streptomycin solution (Gibco, Grand Island, NY, USA). Cells were seeded at a concentration of 2 × 10⁵ in 500 μL of medium using sterile 24-well culture plates. The cells were incubated at 37 °C under a humidified 5% CO₂ atmosphere and treated with DEP (1 mg/mL, 2 mg/mL, and 3 mg/mL). In in vitro study, AM was treated with DEP (1, 2, and 3 mg/mL). After 3 h, the cells were analyzed to investigate the response of AM by DEP in the early stages of inflammation. Furthermore, AM was treated with H₂O₂ (Sigma-Aldrich Corp., St. Louis, MO, USA; 10 mM for 30 min) as a positive control for ROS and was pretreated with antioxidant NAC (Sigma-Aldrich Corp., St. Louis, MO, USA; 5 mM for 2 h).