4.5. Measurement of Cytokines in Tumor Tissue

Tumor tissue samples from each mouse were sonicated at 4 °C in 500–2000 µL of 20 mM Tris–HCl, pH 7.5, 2 mM ATP, 5 mM MgCl₂, and 1 mM dithiothreitol (DTT), and 5 µL of a protease inhibitor cocktail (Sigma)/mL of buffer. They were then centrifuged at 10,000× g for 30 min, and the supernatants were collected for further analysis. A total of three cytokines, namely, IL‑6, TNF‑α, and TGF-β, from tumor tissue homogenates were measured using a quantitative sandwich enzyme technique with Quantikine^® ELISA kits (BD Biosciences), as per the manufacturer’s protocol.