Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

This method can help answer key questions in various metabolic disease research fields about molecular alterations of hepatic energy and protein biosynthesis in obesity, non-alcoholic fatty liver, and type two diabetes. The main advantages of this technique are that it facilitates the isolation of functional primary mouse hepatocytes and detection hepatic nascent proteins via a nonradioactive labeling substrate. Visual demonstration of this method is critical as the perfusion step is difficult to learn because of the small and thin mouse blood vessels.

For liver perfusion, carefully insert a 24 gauge catheter into the Inferior Vena Cava or IVC just at the bifurcation with the right renal vein. Remove the catheter needle maintaining the position of the cannula within the IVC and connect a 24 gauge cannula with perfusion tube by using the connector. Begin perfusing the liver with warm HBSS minus at a four milliliter permanent flow rate quickly cutting the splenic vein to drain out the internal blood.