Lb 940 luminometer

RPTEC cells were seeded into 24-well plates at a density of 1.5 x 10⁵ cells/well. Cells were transfected using Lipofectamine 2000 (ThermoFisher scientific, Waltham, Massachusetts, USA), with 600 ng of DNA and 30 ng of a control vector for transfection efficiency, using the constitutive expression pCMV-Red Firefly Luc Vector (16156, ThermoFisher scientific, Waltham, MA, USA). After transfection, cells were exposed to 10 ng TNFα (< 0.1 endotoxin units per μg of protein, 210-TA, R&D Systems, Minneapolis, MN, USA) or the vehicle control for 24 h and then luciferase activity was determined using a Gaussia-firefly luciferase dual assay kit (16181, ThermoFisher scientific, Waltham, MA, USA) and a LB940 luminometer (Berthold, Germany).

Placental tissue (200 mg) was homogenized. The reactive oxygen species (ROS) concentration analysis was performed by the chemiluminescence (CL) assay using luminol as an indicator of radical formation. The tissue samples were recorded at room temperature using an LB 940 luminometer (Berthold Technologies, Bad Wildbad, Germany) in the presence of enhancers as described previously [16 (link)]. The contents of malondialdehyde (MDA) were assayed using colorimetric methods with a spectrophotometer (Biomate 5, Thermo Electron Corporation, Rochester, NY, U.S.A.). Total antioxidant capacity (TAC) was measured with a commercial kit (Nanjing JianCheng Bioengineering Institute, A015, Nanjing, China) as per manufacturer’s instructions.

We used four HCV subgenomic replicon cell lines, FLR3-1 (genotype 1b, Con-1)47 (link), R6FLR-N (genotype 1b, strain N)26 (link), JFH-1/FLR/K4 (genotype 2a)48 (link), and RMT-tri (genotype 1a)49 (link), which have the firefly luciferase gene for the sensitive and precise quantification of the HCV replication levels using a luciferase assay. We also used REF cells30 (link) which harbor the divided-full genome replicon for analysis of the HCV 5′ UTR sequences. Each cell line was seeded at a density of 5 × 10³ per well in 96-well tissue culture plates, and grown (at 37°C and 5% CO₂) in complete Dulbecco's modified Eagle's medium supplemented with Glutamax I (Invitrogen, Carlsbad, CA) and containing 5% fetal calf serum (Invitrogen). Cells were transfected with 30 nM siRNA using RNAiMax (Invitrogen, Carlsbad, CA). After 72 hours, luciferase activity was determined in triplicate using the Steady-Glo or Bright-Glo luciferase assay kit (Promega Madison, WI). The luciferase signal was measured using an LB940 luminometer (Berthold, Freiburg, Germany) and the results were expressed as the mean percentage of control. IC₅₀ values of siRNA were calculated by nonlinear curve-fitting using the equation: Y = 100-(Y_Bottom × X/(IC₅₀ + X)), where Y represents percent inhibition and X represents the concentration of siRNAs.