Bio plex protm human th17 cytokine assays

Primary cell culture supernatants were analyzed using ELISA kits for IL‐17A (Biolegend or eBioscience), IL‐17F (eBioscience), IL‐2, or IFN‐γ (both from Biolegend). In some cases, cytokine levels were determined by luminex assay (Bio‐Plex Pro^TM Human Th17 Cytokine Assays, Bio‐Rad Laboratories, CA, USA). Fibroblast culture supernatants were analyzed using standard IL‐6 and IL‐8 Biolegend ELISA kits and a luminex assay (R&D) for CXCL1, CXCL5, and CCL20 levels. Cell‐free serum and synovial fluid samples were analyzed using Platinum kits for IL‐17A and IL‐17F (eBioscience) and a luminex assay (Bio‐Plex Pro^TM Human Th17 Cytokine Assays, Bio‐Rad Laboratories) for IL‐1β and IL‐23. All analyses were performed according to manufacturer's instructions.

Supernatants from various culture conditions were collected and stored at −20 °C to analyze cytokines implicated in the Th-17 cell immune response pathway. The Bio-Plex ProTM Human Th-17 Cytokine Assays^® (Bio-Rad Laboratories, Inc., Hercules, CA, USA) were used to evaluate the following cytokines (IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, soluble CD40 ligand (sCD40L), and TNF-α) according to the manufacturer’s recommendations (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Culture supernatants were collected and stored at −80°C until further use. The levels of IL‐23 (eBioscience), IL‐17, IL‐1β, IL‐6, IFN‐γ, IL‐12p70 and TNF‐α (all purchased from BioLegend) were detected using ELISA according to the manufacturers’ protocols. IL‐17A levels in GCF were detected by Luminex assay (Bio‐Plex Pro^TM Human Th17 Cytokine Assays, Bio‐Rad Laboratories, CA, USA).