Cytokine assay

Bead-based assay was conducted to measure the expression level of inflammatory cytokines (TNF-α, IL-6, IL-17A, IL-17F, IFN-γ, IL-2, and IL4) in HCC xenograft tumor tissues. Cytokine beads were purchased from Biolegend (CA, USA). HCC xenograft nude mice were treated with saline, 5-FU, or infliximab, as detailed below. After harvesting from these mice, HCC tumor tissues were cut into small pieces, then minced and filtered for cell precipitation. RIPA buffer with protease inhibitor was added for protein extraction. Subsequently, BCA protein assay was performed to measure the protein concentration. After normalization of protein concentration, the bead-based assay was performed to measure the cytokine levels following the manufacturer’s instructions. Each measurement was repeated 3 times, and the mean of the 3 values was used for final analysis.