2.4. Detection of Caspase Activity in Mouse Liver Tissue

A total of 20–30 mg of liver tissue was homogenized in liquid nitrogen and lysed in 10 mM TRIS-HCl containing 0.5% Nonidet P-40, 10 mM MgCl₂, 150 mM NaCl, 10 mM dithiothreitol and 1% protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). The liver tissue extracts were analyzed for protein concentration using a Lowry assay (BIO-RAD; Munich, Germany) and then diluted in a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM KCl and 5% glycerol for a final concentration of 1 µg/µL. Then, 15 µL extracts were incubated with 15 µL of the caspase substrate DEVD-luciferin and luciferase reagent (Caspase-Glo Promega, Mannheim, Germany) for 1.5 h at room temperature. Following cleavage of the substrate at the DEVD peptide by caspase-3 and caspase-7, aminoluciferin was released, resulting in light production in a luciferase reaction that can be measured in relative light units (RLU) using a luminometer (GloMax^® 96 Microplate Luminometer Mannheim, Germany). Samples were analyzed and compared to the saline treated control group [26].