4.4.5. Enzyme-Linked Immunosorbent Assay (ELISA)

To quantify the release of inflammatory markers from IVD cells, the cell culture medium was collected and analyzed with IL-6 and IL-8 ELISA kit, according to the manufacturer's protocol (IL-6 555220, IL-8 555244, BD Biosciences, San Jose, CA, USA). 96-well plates were coated with capture antibody overnight. After washing, the wells were blocked in assay diluent, washed, loaded with samples or human recombinant IL-6 or IL-8 protein standards, and incubated for 2 h at RT. After washing, detection antibody and streptavidin-horseradish peroxidase (HRP) were applied for 1 h. Next, the plates were washed and substrate solution was added. After 30 min, the reaction was stopped by kit stop solution and the absorbance was measured at 450 nm, with 570 nm correction. The IL-6 and IL-8 concentrations were calculated based on the standard curve. IL-6 and IL-8 in culture media are shown relative to the cytokine treatment.
