Rat tnf α

Rat glioma C6 cell lines provided by American Type Culture Collection (ATCC) were grown in RPMI medium 1640 (Life Technologies) containing 4% fetal bovine serum and 8% horse serum at 37°C in a 5% CO₂ incubator. Three groups were designated as follows: the first was control, the second was pre-treatment with rat TNF-α (10 pg/mL; R&D Systems) for 1 h, and the third was pre-treatment with rat TNF-α for 1 h with etanercept (0.1 μg/ml) added for 30 min. C6 cells were seeded (1 × 10⁶) onto glass slides overnight and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 15 min. The cells were then rinsed twice with PBS and permeabilized with 1% Triton X-100 for 7 min. Next, the cells were pretreated with 1% bovine serum albumin (BSA) in PBS at 25°C for 60 min, incubated with rabbit anti-ADMA polyclonal antibodies (Millipore) at a dilution of 1:1000 for 1 h, and treated with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG polyclonal antibodies at a dilution of 1:10,000 for 1 h. Finally, the cells were washed with PBS, mounted in 90% glycerol containing 4′,6-diamidino-2-phenylindole (DAPI), and photographed under an immunofluorescent microscope (Olympus). In addition, the concentrations of ADMA in the whole cell lysate were examined by ELISA.