Proteome profiler cytokine array kit

A Proteome Profiler Cytokine Array Kit (R&D Systems, Minneapolis, USA) was used on RAW 264.7 cells purchased from ATCC (Wesel, Germany) according to the supplier’s instructions. Cells were either untreated or incubated with 1 µg/mL LPS or 10 µg/mL chitosan (chitosan 70-10) or 1 µg/mL LPS + 10 µg/mL chitosan for 24 h and supernatants were diluted and mixed with the biotinylated detection antibodies. The antibody mixtures were thereafter incubated on a nitrocellulose membrane on which carefully selected capture antibodies were pre-spotted in duplicate. After a washing step to remove the unbound material, streptavidin–HRP and chemoluminescent detection reagents were added sequentially prior to the development of the membrane using an X-ray film for 5 min. The pixel densities were analyzed using image analysis software, Image Lab 3.0.1 (Beta 2) (Biorad Laboratories, Hercules, California, USA), and the corresponding signals on different arrays were compared to determine the relative change in cytokine levels between samples.

The cytokine levels after stimulation with human IL-6 were measured using the Proteome Profiler™ Cytokine Array Kit (R&D Systems, MN, USA). RPMI8226 cells were seeded in 60-mm² dishes in culture medium containing 5% FBS for 24 h. The cells were replenished with serum-free medium for 24 h before treatment with recombinant human IL-6 (10 ng/mL) (R&D Systems, MN, USA) for 18 h. After incubation, all experimental procedures were completed according to the manufacturer's instructions.