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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

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

Published: October 23rd, 2018

DOI:

10.3791/58323

1Department of Pharmacology and Systems Physiology, College of Medicine, University of Cincinnati, 2Division of Endocrinology, Cincinnati Children's Hospital Medical Center, 3Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 4Department of Pediatrics, College of Medicine, University of Cincinnati
* These authors contributed equally

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....

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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

  1. Pre-isolation preparations
    1. Prepare 450 mL of 40% density gradient buffer as described in Table 1 and keep at 4 °C (15 mL/mouse).
    2. Prepare 500 mL of Williams’ Medium as described.......

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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.......

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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.......

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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.

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Name Company Catalog Number Comments
HEPES buffer Fisher Scientific BP310-500
D-glucose Fisher Scientific D16-500
Ethylene glycol-bis(β-aminoethyl ether)-tetraacetic acid AmericanBio AB00505-00025
Antibiotic-Antimycotic (100X) Gibco 15240-062
HBSS (10X) no calcium, magnesium, phenol red Gibco 14185-052
Calcium Chloride Dihydrate (CaCl2.2H2O) Fisher Scientific C79-500
Density gradient buffer GE Healthcare 17-0891-02
DMEM (Dulbecco's Modified Eagle Medium) low glucose, pyruvate Gibco 11885-084
Fetal Bovine Serum Hyclone SH30910.03
Phosphate Buffered Saline (PBS) (1X) Gibco 1897141
Williams medium E, no glutamine Gibco 12551-032
L-alanyl-L-glutamine dipeptide supplement Gibco 35050-061
Collagenase Type X Wako Pure Chemical Industries 039-17864
Perfusion pump Cole-Parmer Masterflex L/S Equipment
IV administration set EXELINT 29081 Equipment
A water bath REVSCI RS-PB-200 Equipment
Tube heater Fisher Scientific Isotemp Equipment
Ethanol Decon Lab, Inc 0-39613
Isoflurane PHOENIX 10250
Autoclaved Cotton Tips Fisherbrand 23-400-124
100 mm Petri Dish TPP 93100
Connector (Male Luer Lock Ring) Cole-Parmer instrument EW-4551807
24G catheters TERUMO Surflo 24Gx3/4'
100 μm Filter (CELL STRAINERS) VWR 10199-658
15 ml conical-bottom centrifuge tubes VWR 89039-666
50 ml conical-bottom centrifuge tubes VWR 89039-658
Chemoselective ligation reaction PROTEIN ANALYSIS DETECTION KIT, TAMRA ALKYNE Invitrogen C33370
AHA (L-azidohomoalanine) Invitrogen C10102
DMEM (methionine free) Gibco 21013024
L-Cystine Dihydrochloride SIGMA C2526
Laemmli sample buffer BioRad 161-0737
Protease Inhibitor Cocktail SIGMA P9599
SDS solution (20%) BioRad 161-0418
Tris-HCL (1M) American Bioanalytical AB14044-01000
Phosphatase Inhibitor Cocktail SIGMA P5726
Protein concentration measuring Kit (Bovin Serum Albumin-BSA) BioRad 500-0207
6-well tissue culture plate TPP 92006
Digital Heatblock VWR 12621-092 Equipment
Multi-Rotator Grant-bio PTR-60 Equipment
Ultrasonic Sonicator Cole-Parmer GE130PB Equipment
Standard Heavy-Duty Vortex Mixer VWR 97043-566 Equipment
A variable mode laser scanner GE Healthcare Life Science FLA 9500 Equipment
Coomassie-dye reagent Thermo Scientific 24594
Inverted microscope Olympus CKX53 Equipment
Western Blotting apparatus BioRad 1658004 Equipment
Centrifuge Eppendorf 5424R Equipment
Automated cell counter BioRad TC20 Equipment
FluorChem R system proteinsimple - Equipment
p-Ampka (T172) antibody Cell signaling 2535
Total-AMPK antibody Cell signaling 5832
Albumin antibody Cell signaling 4929
beta actin antibody Santa Cruz sc-130656
Fine scissors and forceps

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