Cytokine and Protein Analysis in Synovial Fluid

Cytokine and protein concentrations (interleukin- [IL-] 1β, IL-10, aggrecan [ACAN], basic fibroblast growth factor [bFGF], bone morphogenetic protein- [BMP-] 2 and BMP-7, and collagen type 2 cleavage [C2C]) in synovial joint fluids from acutely infected knee joints or coculture supernatants were analyzed by ELISA (RnD, Minneapolis, MN, USA, and BioSource Deutschland GmbH, Solingen, Germany) according to the manufacturers' instructions. The Nitrotyrosine ELISA Kit was purchased from Abcam (Cambridge, UK). Briefly, this assay employs the quantitative sandwich enzyme immunoassay technique. The microplate was precoated with a specific monoclonal antibody. Supernatants were applied to the wells and, after washing, an HRP-conjugated specific antibody was added to the wells. Following the next wash, color development was proportional to the protein concentration and calculated by comparison with a standard. A colorimetric method was applied to quantify total protein amount in the lavage fluids. The bicinchoninic acid (BCA) assay was available in kit form from Pierce (Rockford, IL, USA) and used according to the manufacturer's instructions [4 (link)]. All data from the analyzed cytokines and proteins are reported as relative expression to the total protein content. Statistical calculations were based on these values.