Published: October 23rd, 2018
Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.
Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.
Protein is an important nutritional element and approximately 50% of the dry weight of a human body is composed of proteins which have several biological traits and functions1. Consequently, protein synthesis is one of the most energy consuming events and an alteration in protein metabolism is highly associated with the development of diseases, including metabolic diseases2,3,4. In the liver, protein biosynthesis accounts for approximately 20–30% of total energy consumption5,6. In additio....
This protocol contains the use of laboratory mice. Animal care and experimental procedures were performed according to procedures approved by the animal care committees of Cincinnati Children Hospital Medical Center.
1. Isolation of Primary Mouse Hepatocytes
Primary mouse hepatocytes isolation results in a yield of approximately 20 x 106 total cells/mouse. Histologically, live and attached primary hepatocytes appear polygonal or typical hexagonal in shape with clearly outlined membranous boundary after 24 h incubation (Figure 2).
To confirm whether isolated cells are primary hepatocytes, we compared expression levels of albumin protein in iso.......
Although several immortalized hepatic cell lines have been proposed and used to investigate liver functions49,50,51,52, these cells generally lack the important and fundamental functions of normal hepatocytes, such as the expression of albumin (Figure 3). It is widely recognized, therefore, that utilizing primary hepatocytes is a valuable option for examining liver.......
We thank Drs. Joonbae Seo and Vivian Hwa for their scientific input and discussion. This work was supported by National Institute of Health (NIH) (R01DK107530). T.N. was supported by the PRESTO from the Japan Science and Technology Agency. A part of this study was supported by a grant from NIH (P30DK078392) for the Digestive Disease Research Core Center in Cincinnati.....
|Ethylene glycol-bis(β-aminoethyl ether)-tetraacetic acid
|HBSS (10X) no calcium, magnesium, phenol red
|Calcium Chloride Dihydrate (CaCl2.2H2O)
|Density gradient buffer
|DMEM (Dulbecco's Modified Eagle Medium) low glucose, pyruvate
|Fetal Bovine Serum
|Phosphate Buffered Saline (PBS) (1X)
|Williams medium E, no glutamine
|L-alanyl-L-glutamine dipeptide supplement
|Collagenase Type X
|Wako Pure Chemical Industries
|IV administration set
|A water bath
|Decon Lab, Inc
|Autoclaved Cotton Tips
|100 mm Petri Dish
|Connector (Male Luer Lock Ring)
|100 μm Filter (CELL STRAINERS)
|15 ml conical-bottom centrifuge tubes
|50 ml conical-bottom centrifuge tubes
|Chemoselective ligation reaction PROTEIN ANALYSIS DETECTION KIT, TAMRA ALKYNE
|DMEM (methionine free)
|Laemmli sample buffer
|Protease Inhibitor Cocktail
|SDS solution (20%)
|Phosphatase Inhibitor Cocktail
|Protein concentration measuring Kit (Bovin Serum Albumin-BSA)
|6-well tissue culture plate
|Standard Heavy-Duty Vortex Mixer
|A variable mode laser scanner
|GE Healthcare Life Science
|Western Blotting apparatus
|Automated cell counter
|FluorChem R system
|p-Ampka (T172) antibody
|beta actin antibody
|Fine scissors and forceps
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