Preparation of Rat Liver Microsomes

Rats were killed by cervical dislocation after they were starved for 12 h. The liver of the rat was exposed by opening the abdominal cavity and was perfused with 0.15 mol/L KCL buffer (pH = 7.4) through the superior vena cava till the blood was flushed off. Precisely 3 g liver tissue was removed and transferred to a petri dish and kept at 0–4°C. The tissue was homogenized in 0.15 mol/L KCL-PBS buffer (stored at 4°C). The homogenate was centrifuged (Optima XPN, Beckman Coulter, USA) at 9000 × g for 30 min at 4°C, and the supernatant, thus obtained, was precipitated by centrifugation at 105,000 × g for 60 min at 4°C. The precipitate containing liver microsomes was removed, and resuspended in 0.15 mol/L KCL-PBS buffer. The centrifugation (105,000 × g for 60 min at 4°C) and resuspension (0.15 mol/L KCL-PBS buffer containing 0.25 mol/L sucrose) steps were repeated for the preparation of liver microsome homogenate, which was then stored at −80°C until use. The concentration of liver microsomes was 8.838 mg/mL, as determined using the BCA protein quantitation kit.