Mouse tnfα elisa

Pam2.12 cells were seeded in 96-well dish and stimulated with 250 μM NiCl₂ for 0, 12, 24, 48, 72 h before analysis of Sema3A production in medium using a mouse Sema3A ELISA kit (Signalway Antibody, College Park, MD, USA). Pam2.12 cells seeded in 96-well dish was performed with Sema3A siRNA transfection using INTERFERin as described above to suppress Sema3A expression. Culture supernatant 24 h after NiCl₂-stimulation was collected and stored at −20 °C until analysis. The TNF-α production was measured using a mouse TNF-α ELISA (eBioscience, San Diego, CA, USA), according to the manufacturer instructions.

Blood was centrifuged at 800 g for 10 minutes, and the supernatants were collected for TNF-α analysis in plasma. ELISA was performed as recommended by the manufacturer (mouse TNF-α ELISA, eBioscience Inc, San Diego, USA). Briefly, to a 96 well plate pretreated with coating buffer was added 100 μl of capture antibody (anti-mouse TNF-α) in coating buffer. The plate was sealed and incubated overnight at 4°C. Wells were aspirated and washed 5 times with 200 μl of a washing buffer (0.05% Tween-20 in phosphate buffered saline (PBS)) and were blocked with 200 μl of assay diluent solution (10% fetal bovine serum in PBS). After incubation at room temperature for 1 hour, 100 μl of standard recombinant mouse TNF-α along with the samples were added to the appropriate wells, and the wells were incubated at room temperature for 2 h. Then, 100 μl of detection antibody (anti-mouse TNF-α) in assay diluents were added to each well, and the wells were incubated at room temperature for 1 h. Finally, 100 μl of avidin-horseradish peroxidase (HRP) diluted in assay diluents were added, and the wells were incubated at room temperature for 30 min to activate the substrate solution. The reaction was stopped by using 2N H₂SO₄. Values of the optical density of the samples were read at 450 nm by using a spectrophotometer (Thermo Max Microplate Reader, Molecular Devices LLC, Sunnyvale, USA).