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

Summary

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.

Abstract

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.

Introduction

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 diseases^2,3,4. In the liver, protein biosynthesis accounts for approximately 20–30% of total energy consumption^5,6. In additio....

Protocol

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 in Table 1 and keep at 4 °C.
   3. Prepare 500 mL of DMEM as described in Table 1 and keep on ice (30 mL/....

Results

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.......

Discussion

Although several immortalized hepatic cell lines have been proposed and used to investigate liver functions^49,50,51,52, 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.......

Materials

List of materials used in this article

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
24 G 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