Dulbecco s minimum essential medium

Hep3B, HepG2, and Huh7 cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in Dulbecco’s minimum essential medium (Corning, NY, USA), supplemented with 10% fetal bovine serum (FBS; Corning) and 1% penicillin–streptomycin (P/S; Corning). Human NK-92 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in α-minimum essential medium (Corning), which was composed of 12.5% FBS (Corning), 12.5% horse serum (HS; Sigma-Aldrich, St. Louis, MO, USA), 1% P/S (Corning), 0.2 mM Myo-inositol (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich), 0.02 mM folic acid (Sigma-Aldrich), 10 ng/mL Interleukin (IL)-2 (Miltenyi Biotec, Bergisch Gladbach, Germany), and 20 ng/mL IL-15 (Miltenyi Biotec) activating cytokines. Normal human liver THLE-2 cells were obtained from Dr. KP Kim (Asan Medical Center, Seoul, Korea) and were cultured in bronchial epithelial cell growth medium (BEGM Bullet Kit; Lonza, Basel, Switzerland) as per the manufacturer’s protocol. All cells were cultured at 37 °C with 5% CO₂.

The human neuroblastoma cell line SH-SY5Y (ATCC, USA) was cultured in Dulbecco’s minimum essential medium (Corning, New York, NY, USA) supplemented with glutamine and antibiotics. Differentiated SH-SY5Y cells are broadly used for in vitro studies involving neuronal-like cells. Differentiated SH-SY5Y cells are low cost to culture, and the ethical concerns related to primary human neuronal culture are avoided. Moreover, since SH-SY5Y cells are human-derived, they present numerous human-specific proteins and isoforms that would not be intrinsically present in rodent primary cultures [23 (link)]. To induce the differentiation, SH-SY5Y were plated at 15,000 cells/cm² and, after 24 h, were grown in 10 μM Retinoic acid of RA with 1% FBS for 3 days. After 3 days, the media was replaced with fresh 12-O-tetrade-canoyl-phorbol-13-acetate (TPA) (Sigma, St. Louis, MO, USA) for another 3 days of differentiation. For the setup of the in vitro PD model, the differentiated SH-SY5Y cell line was treated with 6-hydroxydopamine (6-OHDA) (Sigma) for 24 h after differentiation. SCMC and S-methyl-L-cysteine sulfoxide (SCMC-O) treatments were performed one hour before 6-OHDA stress and used at the final concentration of 0.25 mM while NAC was performed at the final concentration of 5 mM according to Yakamuro et al. [24 (link)].