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.
After 10 minutes, perfuse the mouse liver with 35 milliliters of HBSS plus supplemented with Collagenase Type X at a flow rate of four milliliters per minute, clamping the splenic vein for about 10 seconds every few minutes to facilitate distribution of the perfusate throughout the liver. When all of the Collagenase has been perfused, transfer the liver onto a lab tissue before stopping the pump to avoid blood backflow into the liver. Use forceps to remove the gallbladder and gently wipe the liver with a fresh lab tissue to remove any blood.
Place the liver in a 100 millimeter Petri dish containing five milliliters of fresh warm HBSS plus supplemented with Collagenase and use stray-tipped forceps to gently remove the liver capsule. Use the forceps to carefully disperse the parenchymal tissue and add 15 milliliters cold DMEM to the Petri dish. Shake the torn liver gently to release the residual parenchymal cells into the medium and add another 15 milliliters of cold DMEM to the dish to acquire the rest of the cells.
Then filter the tissue slurry through a 100 micron cell strainer into a 50 milliliter conical tube on ice. To isolate the primary mouse hepatocytes, collect the cells by centrifugation and resuspend the pellet in 10 milliliters of fresh DMEM. Carefully layer the cells over a 40%density gradient buffer for density gradient separation, discarding the cells from the upper and middle layers after centrifugation.
Resuspend the primary parenchymal hepatocyte pellet with two to three milliliters of William's Medium E to avoid collecting dead cells from the wall of the tube and transfer the cells into a new 50 milliliter tube. Mix the cells with an additional seven to eight milliliters of William's E Medium before centrifugation and resuspend the pellet in 10, 20, or 30 milliliters of fresh William's Medium E depending on the size of the pellet. After counting, seed six times 10 to the fifth cells to each well of a six-well plate for a two to three-hour incubation at 37 degrees Celsius.
At the end of the incubation, replace the medium with DMEM supplemented with 10%Fetal Bovine Serum and anti-anti to remove the dead and unattached cells and return the cells to the incubator overnight. For azide-modified nascent protein labeling, add 20 to 40 micrograms of cell lysate to 50 microliters of 2X reaction buffer. Bring the volume to 80 microliters with distilled deionized water and vortex the protein solution for five seconds.
Add five microliters of copper sulfate to the cells for another five-second vortex, followed by five microliters of reaction buffer additive one with five seconds of vortexing. After a three-minute incubation, add 10 microliters of reconstituted reaction buffer additive two to the cells for another five-second vortex and rotate the sample for 20 minutes at four degrees Celsius on a multi-rotator machine protected from light. At the end of the rotation, add 300 microliters of methanol to the lysate, followed by 75 microliters of chloroform, and 200 microliters of double distilled water.
Collect the labeled protein by centrifugation and add 250 microliters of fresh methanol to the pellet. After vortexing, centrifuge the lysate again and cover the bare pellet with a lint-free tissue for 15 minutes at room temperature. For PAGE analysis, solubilize the dried pellet in 20 microliters of loading buffer without beta-mercaptoethanol and vortex the pellet for 10 minutes.
Next, heat the protein in a 70 degree Celsius heat block for 10 minutes and load the sample onto a 10%sodium dodecyl sulfate gel for tetramethylrhodamine detection. Then use a variable mode laser scanner for the precise quantitation of the AHA-labeled nascent proteins. Histologically, live and attached primary hepatocytes appear typically polygonal or hexagonal in shape with a clearly outlined membranous boundary after 24 hours of culture.
To confirm whether the isolated cells are primary hepatocytes, in this experiment, albumin protein expression levels were compared in mouse embryonic fibroblasts, a mouse hepatoma cell line, isolated primary mouse hepatocytes, and mouse livers. The albumin protein expression was detected in primary mouse hepatocytes and mouse livers, but was not detectable in either mouse embryonic fibroblasts or hepatoma cell line cells. Primary hepatocyte treatment with 10 micromolar Rotenone for four to six hours revealed a robust induction of hepatic AMP-activated protein kinase as detected by increased phosphorylation levels on hepatic AMP-activated protein kinase T172 similar to those observed in vivo.
Indeed, five hours of 10 micromolar Rotenone treatment had profound inhibitory effects on nascent protein synthesis in treated primary hepatocytes compared to untreated control cells as demonstrated by a chemoselective ligation reaction assay. While attempting this procedure, it's important to remember to prepare all required reagents and mediums in advance. This technique paved the way for the researcher in the field of hepatic metabolic pathophysiology to explore the molecular mechanism of energy protein regulation in obesity and type two diabetes in isolated primary mouse hepatocyte.
