Human tnf alpha elisa ready set go kit

Generally, all chemicals were obtained from FUJIFILM-Wako (Tokyo, Japan), unless stated otherwise. Fetal bovine serum (FBS) for cell culture was purchased from Gibco (GIBCO BRL, Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8) and Cytotoxicity lactate dehydrogenase (LDH) Kit-WST were purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Cellular ROS Kit (#ab113851) was obtained from Abcam (Tokyo, Japan). Nuclear Extraction (NE)-Kit and human TNF-alpha ELISA Ready-SET Go Kit (#88-7324) were obtained from eBiosciences (San Diego, CA, USA), respectively. Pierce bicinchoninic acid-(BCA) protein Kit and Enhanced chemiluminescent-(ECL) Western blotting substrate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Bovine serum albumin-(BSA) fraction V was purchased from Roche (Mannheim, Germany). All antibodies for Western blot analysis were purchased from Cell Signaling Technology (Tokyo, Japan), except anti-Nrf2 (Proteintech Corp., Tokyo, Japan), anti-p65 NF-ĸB, and horseradish peroxidase (HRP)-conjugated second antibody (Sigma-Aldrich, Burlington, MA, USA), anti-rabbit (IgG) Alexa Fluor^TM 488-conjugate (Alexa-488), anti-rabbit (IgG) Alexa Fluor^TM 594-conjugate (Alexa-594) (Molecular Probes, Invitrogen, CA, USA). DAPI (4′,6-diamino-2-phenylindole) dye used for the nuclei staining was purchased from Molecular Probes (Eugene, OR, USA).

PBMC (1 × 10⁶ cells/ml) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100U/ml penicillin/streptomycin at 37°C in a humidified incubator with 5% CO₂. Cells were treated with solvent control (0.01% DMSO) or 100 μM goitrin for 72 h w/wo addition of 20 IU/ml IL-2. Supernatants were used for photometric quantification by the human TNF-alpha ELISA Ready-Set-Go kit from eBIoscience (Frankfurt, Germany) according to the manufacturer's instructions.