4.7. Enzyme-Linked Immunosorbent Assay (ELISA) for TNF-α and IL-6

Liver (approximately 30 mg) was homogenised in lysis buffer (137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1% NP40, 10% glycerol, 1 mM PMSF, 10 μg/mL aprotinin, 1 μg/mL leupeptin, 0.5 mM sodium vanadate, Sigma-Aldrich, Dublin, Ireland) and centrifuged at 13,000× g at 4 °C for 15 min. Protein concentration was determined in supernatant using the Bradford assay. TNF-α and IL-6 were evaluated using specific commercially available ELISA kits according to supplier’s protocol (PeproTech, NJ, USA). Absorbance was read at 450 nm and cytokine levels in liver tissue are expressed as ng/g protein.