Measuring Cytokines in Murine Arthritis

Cytokine measurements were performed on samples of subcutaneous flush fluid acquired from hind paws as described by Kovács et al. (2014) (link). Briefly, after induction of arthritis, on day 3, mice were anesthetized with ketamine/xylazine (100/10 mg kg⁻¹) and were sacrificed via cervical dislocation. Subcutaneous tissue of the hind paws was flushed immediately with 1 mL of ice-cold PBS solution supplemented with 10 mmol L⁻¹ of EDTA (pH 7.5) and 20 mmol L⁻¹ of HEPES (pH 7.4). Samples were then snap frozen in liquid nitrogen and were stored at −80°C. Quantitative determination of IL-1β, KC, MIP-1α, and MIP-2 concentrations from the samples were performed using MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel—Immunology Multiplex Assay (Merck Millipore, Billerica, MA, USA). A Luminex 100 device was used for the measurement, and the interpretation of data was performed with the Luminex 100 IS software.