ELISA

Sera and joint fluid were harvested from all animals 10 days after the induction of arthritis and kept frozen until biochemical analysis. Multi‐Analyte ELISArray kits (Qiagen, Valencia, CA, USA) designed for mice were used for the quantitative measurement of 12 different cytokines and chemokines (e.g. IL‐1β, IL‐6, TNF‐α, VEGF and IL‐17A). For the serum fluid, a total of five measurements per treatment were made and the graphs show the mean with standard deviation. The joint fluid was collected by cutting open capsules of ankle joints and by washing the joint cavity with 5 μl PBS, and 1 μl of withdrawn joint fluid was diluted 10‐fold to assess the concentrations of cytokines/chemokines. The synovial fluid measurements were made by combining the samples of a pool of five mice per group. We read the absorbance at 450 nm within 30 min of stopping the reaction. Standards were used, standard curves were plotted and the experimental protein values were calculated. Results were expressed as cytokine/chemokine fold increases, calculated as the ratio between protein levels in HMGB1‐, BoxA‐, sFlt‐1‐treated mice and control CAIA mice. The measurement was performed only once, and therefore a standard deviation was not determined.