Interleukin 6 (il 6)

The levels of cytokines and growth factors in the hucMSC conditioned medium were assayed using ELISA kits (VEGF, HGF, NGF, and KGF, Jiamay Bitotech; IL-6, IL-8, and TGF-β1, Neobioscience) following the manufacturer's protocol.

The concentrations of IFN-γ, IL-4, IL-6, IL-10, and IL-17 (Neobioscience, Shenzhen, China) in the spleen CD4⁺ T cell culture supernatants or serum of mice were determined by commercially available ELISA kit following the manufacturer's instructions.

Assays for proinflammatory markers were carried out by means of commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions; ELISA kits for the following molecules were used: human IL-1β, IL-6, and TNF-α (all from Neobioscience, Shenzhen, China). First, the cell supernatants were added to microplate wells, which were precoated with specific antibodies in advance. After 1.5 h of incubation at 37°C, the unbound supernatants were washed away, and enzyme-linked antibodies were added to each well, followed by incubation for 1 h at 37°C. Then, a second washing step was performed to remove the unbound reagents. Finally, the enzyme-binding diluent was pipetted into each well, followed by incubation for 30 min, and the stop solution was added to end the color development reaction. The absorbance of the samples at 450 nm was measured using the microplate reader. A standard curve based on the results obtained using eight human standard solutions was plotted, and the sample concentrations were confirmed. All assays were performed in triplicate for each experiment.

Intracellular AGE levels and inflammatory cytokines were assessed by ELISA kits specific to AGEs (Sigma‐Aldrich), TNF‐α, IL‐1β and IL‐6 (Neobioscience, Shenzhen, China) according to the manufacturer's instructions.

Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum and the antibiotic-antimycotic solution were purchased from Gibco (Auckland, New Zealand). The ELISA kits for TNF-α, IL-1β and IL-6 were obtained from Neobioscience (Beijing, China). Primary antibodies against extracellular signal-regulated kinase (ERK), phospho-ERK, p38, phospho-p38, c-Jun N-terminal kinase (JNK), phospho-JNK, IκB-α, phospho-IκB-α, p65, phospho-p65, IKKβ and phospho-IKKβ were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated secondary antibodies were also obtained from Cell Signaling Technology.