Penicillin g

HepG2 cell line was purchased from ATCC and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium in supplement with 10% fetal bovine serum (FBS), penicillin G (100 U/mL), streptomycin (100 μg/mL). All cell culture supplies were obtained from Gibco (Waltham, MA). HepG2 cells were treated with PBS (vehicle control) or different concentration of SS (25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL and 400 μg/mL) for 12 h or 24 h. At the end of treatment, images of cells were taken. Cell viabilities were determined using Cell Counting Kit-8 (Dojindo, D.C. USA), according to manufacturer’s instruction. Intracellular ROS, GSH levels and iNOS activity were determined, as described above (Method 2.6 Redox status assessment).

Immortalized mouse brain endothelial cells (bEnd3) from ATCC (Manassas, VA, USA) were used as a representative model for a mouse BBB endothelium. bEnd3, passage 25–35, were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/mL) and streptomycin (100 μg/mL); all were purchased from ATCC (Manassas, VA, USA). Cultures were maintained in a humidified atmosphere (5% CO₂) at 37 °C incubator (VWR International, Radnor, PA, USA).

Human skin fibroblasts (AG01518; passages 12–24; Coriell Institute, Camden, NJ, USA), malignant melanoma cells SK-MEL-28 (HTB-72; ATCC, Manassas, VA, USA) and neuroblastoma cells SH-SY5Y (94030304; Sigma-Aldrich, St. Louis, MO, USA) were cultured in Eagle's minimum essential medium (EMEM) GlutaMAX, supplemented with 50 IU/ml penicillin-G, 50 μg/ml streptomycin, and 10% fetal bovine serum (all from Gibco, Paisley, UK). Melanocytes were kindly provided by Petra Wäster and cultured as described previously (Andersson et al., 2001 (link)). Human cervical cancer cells HeLa (CCL-2; ATCC), breast cancer cells MDA-MB-231 (HTB-26; ATCC) and human colon cancer HCT-116 (CCL-247; ATCC) were cultured in Dulbecco's modified eagle medium GlutaMAX supplemented with 50 IU/ml penicillin-G, 50 μg/ml streptomycin and 10% fetal bovine serum. Cells were incubated in humidified air with 5% CO₂ at 37°C. The day before experiments, cells were trypsinized and seeded to reach 50% confluence. For microscopical examination cells were seeded on glass coverslips No 1.0.