Raw 264.7 cells

RAW264.7 cells (TIB-71, ATCC, Manassas, VA, USA) were obtained from American Type Culture Collection (ATCC). NO production was measured using a previously described method [43 (link),44 (link)]. Briefly, RAW264.7 macrophages were seeded into 96-well plates at a density of 5 × 10⁵ cells/well, and after being incubated with samples for 1 h, the cells were treated with LPS (0.1 µg/mL) for 24 h. Equal volumes of the cell culture supernatant and Griess reagent were mixed, and the absorbance of the mixtures was recorded at 550 nm. The levels of PGE₂, IL-1β and IL-6 cytokines were measured with ELISAs. RAW264.7 cells (2 × 10⁶ cells/well) were cultured in a 6-well plate for 24 h. The cells were activated by LPS (0.1 µg/mL) for 16 h after being pretreated with samples for 1 h, and the concentrations of PGE₂, IL-1β and IL-6 in the supernatants were measured using an ELISA kit (R&D Systems, Minneapolis, MN, USA). The cell viabilities were quantified with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay after 24 h of incubation with the samples [45 (link)].

Murine monocyte/macrophage RAW 264.7 cells (Sumitomo Dainippon Pharma, Osaka, Japan) were grown in minimal essential medium alpha (Nacalai Tesque) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Biowest, Nuaille, France), 100 U/mL penicillin, and 100 μg/mL streptomycin (Wako Pure Chemical Industries, Osaka, Japan) at 37°C in a humidified atmosphere of 5% CO₂ in air. For differentiation of osteoclasts, RAW 264.7 cells (5,000 cells per well in a 96-well plate) were cultured in the presence of 10 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL; R&D Systems) for 2 days, and then the cells were stimulated with 10 ng/mL RANKL and 10 ng/mL recombinant human TNF-α (R&D Systems) in the presence or absence of the drugs for 3 days.

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.