Spleen and hind paws were harvested from water-fed arthritic control rats and TCE-fed rats at peak phase of adjuvant arthritis. Single-cell suspensions from the spleens and synovial-infiltrating cells (SIC) from the joints were prepared and stimulated under in vitro conditions for 6 h with 5 μg/mL phorbol 12-myristate 13-acetate (PMA) (Promega) and 100 μM ionomycin (Sigma-Aldrich) in the presence of 100 μg/mL Brefeldin A (Life Technologies) for the final 4 h of culture.25 (link) Cells were surface-stained with anti-CD3 APC and anti-CD4 FITC (all from eBioscience). Thereafter, cells were fixed and permeabilized using a BD Fixation/Permeabilization Kit (BD Bioscience) and stained for intracellular cytokines using anti-IL-17A eFluor 450 (eBioscience) and anti-IFN-γ PE (Biolegend), and then analyzed on an LSRII flow cytometer (BD Biosciences). The data were analyzed with FlowJo Software (TreeStar).