Cytokine quantification by ELISA

Concentrations of cytokines in the supernatants of cultures were quantified using a DuoSet ELISA kit (R&D System, Minneapolis, MN, USA) following the manufacturer’s instructions with slight modification. Briefly, 96-well plates were coated with monoclonal antibodies at 4 °C overnight. Standards or samples were applied to individual wells and incubated at 4 °C overnight followed by washing and the application of biotinylated antibodies for 1 h at room temperature. After washing, HRP–streptavidin conjugate was added for 1 h at room temperature followed by detection with TMB (Sigma). The reaction was stopped with 1 M H₂SO₄ and quantified at 450 nm with a microplate reader (Bio-Rad, Hercules, CA, USA).The limits of detectability for human cytokine assays were as follows: IL-1ß, 3.91 pg/mL; IL-2, 25 pg/mL; IL-10, 31.2 pg/mL; IL-12, 31.2 pg/mL; TNF-γ, 15 pg/mL, and IFN-α, 7.8 pg/mL. Serum VIM levels wee examined by a double antibody sandwich ELISA (Human Vimentin (VIM) ELISA Kit, CUSABIO, Catalog Number. CSB-E08982h). ELISA was performed in duplicate, and other assays were performed in strict accordance with the manufacturers’ instructions.