Inflammatory cytokine assays

The levels of pro-inflammatory cytokines (IL-1β, TNF-α) and anti-inflammatory cytokines (IL-4, IL-10) were quantitatively detected by the indirect ELISA technique [47]. Briefly, 50 μL of protein samples containing the same amount of proteins were prepared using bicarbonate/carbonate coating buffer (Sigma-Aldrich). Samples were loaded in a PVC ELISA microplate (Corning), sealed, and incubated overnight at 4°C. After four washes, the remaining protein-binding sites in the coated wells were blocked by adding 200 μL blocking buffer (1% BSA in PBS, 0.3% solution of H₂O₂) for 1 h at room temperature. Afterwards, 50 μL of specific antibodies were added and incubated for 4 h at 37°C. The plate wells were then washed and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the plate was washed and developed by incubating with TMB (3,3′,5,5′-tetramethylbenzidine) solution (Thermo Fisher) for 30 min, and the reaction was stopped with 50 μL of sulfuric acid. The plate wells were then read at 450 nm on a spectrophotometer (Bio-Rad), and values were calculated and expressed as percent change versus control group.