Complete dulbecco s modified eagle medium

Human hepatocellular carcinoma HepG2 (ATCC), Huh7 (provided by Dr. Zhong-Zhe Lin, National Taiwan University Hospital, Taipei, Taiwan), Huh7R (Dr. Zhong-Zhe Lin), and colorectal cancer HT29 (ATCC) cells were maintained in complete Dulbecco’s Modified Eagle Medium (GIBCO BRL, Gaithersburg, MD, USA) or Roswell Park Memorial Institute 1640 Medium (no HEPES; GIBCO BRL) containing 10% fetal bovine serum (Biological Industries Ltd., Kibbutz Beit Haemek, Israel; Invitrogen, Carlsbad, CA, USA) and placed in an incubator at 37 °C with a humidified atmosphere of 5% CO₂. The curcumin dissolved in dimethyl sulfoxide was stored at −20 °C. Before the experiments, the curcumin stock was diluted to the final indicated concentrations with complete Dulbecco’s Modified Eagle Medium; control cells were cultured in a medium containing an equal amount of dimethyl sulfoxide without curcumin.

The VN titer was measured in eight randomly selected sera from the germanium biotite and seven sera from the control groups of Farm B at 20 and 31 weeks after vaccination (Tables 1 and 3). Fifty microliters of twofold serially diluted bovine sera were reacted with 50 μl of the FMDV Andong strain (100TCID₅₀), a serotype O FMDV Korean isolate from the 2010 outbreak, at 37°C for 1 h. After incubation, 50 μl of complete Dulbecco’s Modified Eagle Medium (Gibco, USA) with 5% fetal bovine serum containing 1 × 10⁴ LFBK cells/ml was added to the reaction mixture. After incubation of the reaction mixtures at 37°C for 48 h, the VN titer was determined as the final dilution of serum showing no cytopathic effect.

Duck primary myoblasts were extracted from the pectorales of E13 Shan Ma duck embryos (Tianyun). The breasts of E13 ducks were collected and washed in pre-cooled phosphate-buffered saline with 0.5% penicillin/streptomycin (Invitrogen). The muscle samples were cut into pieces using scissors and then trypsinized (Gibco, Grand Island, NY, USA) at 37 °C for 40 min after the skin and bones had been removed. The digestion was terminated with fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). The mixture was filtered using a 40-μm cell strainer (Sangon, Shanghai, China) and centrifuged at 1100 rpm for 5 min. After serial plating, the cells were grown in complete Dulbecco’s modified Eagle medium (Gibco, Grand Island, NY, USA) with 15% FBS and 0.2% penicillin/streptomycin and incubated at 37 °C in a 5% CO₂ humidified atmosphere. The myoblasts were induced to differentiate using 0.5% FBS. A total of 5 µg of DNA plasmid and 5 nmol of siRNA or siRNA NC were mixed with 5 × 10⁶ myoblasts in 200 μL of Entranster-E electroporation solution (Engreen, Beijing, China), and cell electroporation was performed on a BTX ECM2001 (BTX, San Diego, CA, USA).