Hmgb1

Male and female mice at 7, 12, and 16 months of age were anesthetized with isoflurane gas 4%, and blood was collected from the retro-orbital sinus. Blood samples were incubated for 2h at room temperature for clotting and then centrifuged at 1,800 × g for 5 min twice. The serum samples were aliquoted and stored at −80°C until analysis. The detection of inflammatory mediators in serum was performed by using commercial enzyme-linked immunosorbent assay (ELISA) kits: LCN2 (Abcam, #ab199083), CXCL10 (Abcam, #ab260067), CCL5 (Abcam, #ab100739), C3 (Abcam, #ab157711), S100b (Abcam, #ab234573), CCL20 (Abcam, #ab100728) and HMGB1 (Cloud-Clone Corporation #SEA399). Samples were diluted 1/2 – 1/3 and analyzed by replicates following the manufacturer’s instructions. Mouse cytokine arrays were performed using the Proteome Profiler Mouse XL cytokine array kit (R&D systems, #ARY028), which contains 111 captured antibodies against inflammatory mediators, in two samples from 16 month-old mice of each genotype. Cytokine profiles were performed as instructed in the manufacturer’s directions. Membrane quantifications were performed with HLImage software (Western Vision Software).