Human tnf duoset

The concentration of tested cytokines (TNF, IFN-γ, IL-10, IL-35 and TGF-β) was detected in culture supernatants using a specific ELISA. The secretion of TNF was evaluated using Human TNF DuoSet (R&D Systems, Minneapolis, MN, USA); that of IFN γ, IL-10 and TGF-β by Human ELISA Ready-SET-Go!® (eBioscience); and that of IL-35 concentration by ELISA Kit for IL-35 (Uscn Life Science Inc., Wuhan, People’s Republic of China). Optical density was determined with an ELISA reader at 450 nm, and the detection range was 15.6–1000 pg/mL for TNF, IL-35 and TGF-β, whereas it was 3.6–500 pg/mL for IFN-γ and 4.4–300 pg/mL for IL-10.

The supernatant levels of IL-10 and TNF-α were measured using human IL-10 DuoSet (R&D Systems) and human TNF DuoSet (R&D Systems) according to the manufacturer’s instructions.

Human TNF duo set (R&D System) was used to quantify TNF as per the manufacturer recommendations. Briefly, 96-well plates were preincubated with TNF capture antibody for 16 h followed by blocking with 1% FBS. TNF (1–1000 pg/ml) was used as standards. The samples were added to the plates for 16 h followed by the detection antibodies for another two h. Next, 100 µL/well of substrate solution was added. The enzymatic reaction was stopped with 50 μL/well of stop solution (BioFX Labs, Owing Mills, MD). The plates were read at 490 nM using iMark Microplate reader (Biorad, Mississauga, Ontario) using microplate manager 6 software.