species, such as hydroxyl radicals, singlet oxygen radicals, nitrogen oxide, and nitrogen, were 20142041 confirmed by optical emission spectroscopy. We exposed above NEAPP to RPMI-1640 separately from the cells, which is designated `Nonequilibrium atmospheric pressure plasmas activated medium ‘. The separated distance between the plasma source and medium is critical to consistently reproduce data, and so all experiments were performed under the same conditions, L = 15 mm, where no plasma discharge were contacted with the medium. The duration of plasma treatment was ranged from 0 to 300 seconds in vitro studies. Six mL of RPMI-1640 medium was placed in 60-mm dish. The center on each 60-mm dish was treated for several exposure times with NEAPP, indicated by NEAPP-AM-30, -60, -120, -180 and -300 respectively below. For animal treatment, four mL of medium was placed in 21-mm dish and was treated with NEAPP for 600 sec. Materials and Methods Cell culture The NOS2 and NOS3 cells, derived from serous EOC, were established in our institute. These cell lines were maintained in RPMI-1640 supplemented with 10% fetal Bovine serum and penicillin-streptomycin at 37uC in a humidified atmosphere of 5% CO2. Chemosensitivity assay The paclitaxel/cisplatin chemo-sensitivity assay was performed as described previously. Briefly, cells were seeded in triplicate in 96-well plates at a density of 2,000 cells in a volume of 100 mL of culture medium containing 10% FBS. After incubation for 24 hrs at 37uC, the medium was replaced with fresh medium with or without various concentrations of paclitaxel and cisplatin. After an additional 72 hr, cell viability was assayed using the Aqueous One Solution Cell Proliferation Assay kit, according to 
the manufacturer’s instructions. MedChemExpress Luteolin 7-O-β-D-glucoside Absorbance was then measured at 490 nm with a microplate reader. IC50 values indicate Establishment of paclitaxel/cisplatin-resistant EOC cells The NOS2TR and NOS2CR cells, established from parental NOS2 cells in our institute, had acquired chronic Plasma Therapy for Chemoresistant Ovarian Cancer the concentrations resulting in a 50% reduction in growth as compared with control cell growth. Reactive oxidative species inhibition and L-c-glutamyl-L-cysteinyl-glycine depletion To inhibit ROS, N-acetyl cysteine, an intracellular ROS scavenger, was used. In addition, L-buthionine–sulfoximine is an inhibitor of GSH synthesis. It is known that GSH is the most abundant and effective component of the defense system against free radicals including ROS. The compounds NAC and BSO were added to cells at a final concentration of 4 and 2 mM in PBS, respectively. The required volume of each drug was added directly to complete cell culture medium 2 hrs before NEAPP-AM treatment and NEAPP-AM to achieve the desired final concentrations, respectively. Cell viability was examined with the “Cell viability assay”. Cell viability assay The effect of NEAPP-AM on the viability of cells was determined by the Aqueous One Solution Cell Proliferation Assay kit described in “Chemosensitivity assay”. The cells were plated in 96-well plates at a density of 16104 cells per well in 100 mL of complete culture 15102954 medium. The next day, cells were treated with NEAPP-AM for 24 hrs, and the above conditions were optimized to detect the NEAPP-AM sensitivity of the cells. Each activated time for NEAPP-AM was repeated in 6 wells. Experiments were performed in triplicate. 3 Plasma Therapy for Chemoresistant Ovarian Cancer Cell apoptosis assay/casp
