2.8. Measurement of cytokines using Enzyme-linked immunoassay (ELISA)

The concentration of IL-6 and TNF-α in serum were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R & D systems, Minneapolis, MN, USA). Assays were performed following the manufacturer’s instructions. Briefly, a 10× diluted serum or 1.5 mg/ml tissue protein along with known concentrations of cytokine standards were applied to the primary antibody pre-coated 96-well ELISA plates. After overnight incubation at 4 °C, a biotinylated secondary antibody was applied and incubated for one hour at room temperature. After incubation, streptavidin-horseradish peroxidase (SAHRP) solution was applied, followed by tetramethylbenzidine (TMB) substrate solution. Concentrations of the cytokines were estimated from the absorbance at 450 nm using a spectrophotometer (SpectraMax M2e Multi-Mode Microplate Reader, Molecular Device, LLC, San Jose, CA, USA). Protein concentrations in the tissue lysates were quantified using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).