Name: isolation of primary mouse hepatocytes for nascent protein synthesis analysis by non-radioactive l-azidohomoalanine labeling method
Uploaded: 2018-10-23T13:30:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method at JoVE.com

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.

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
<ol>
	<li>Pre-isolation preparations
	<ol>
		<li>Prepare 450 mL of 40% density gradient buffer as described in Table 1 and keep at 4 &#176;C (15 mL/mouse).</li>
		<li>Prepare 500 mL of Williams&#8217; Medium as described in Table 1 and keep at 4 &#176;C.</li>
		<li>Prepare 500 mL of DMEM as described in Table 1 and keep on ice (30 mL/mouse).</li>
		<li>Prepare 500 mL of HBSS (-) Buffer as described in Table 1 and keep at 42 &#176;C in the water bath (40 mL/mouse).</li>
		<li>Prepare 500 mL of HBSS (+) Buffer with 0.3 mg/mL Collagenase Type X as described in Table 1 with 30 min incubation at 42 &#176;C in the water bath (40 mL/mouse).</li>
		<li>Set up the perfusion pump by placing the intravenous (I.V) administration tubing across the latching foot pedal and by checking the flow of flushing solution without air bubbles.</li>
		<li>Warm up the tube heater at 42 &#176;C.</li>
		<li>Cool down the centrifuge machine to 4 &#176;C.</li>
	</ol>
	</li>
	<li>Anesthesia and Perfusion
	<ol>
		<li>Clean the work areas and the pump.</li>
		<li>Rinse out the pump-connected IV tube by running 70% ethanol thoroughly for 10 min.</li>
		<li>Remove the residual ethanol from the pump-connected IV tube by rinsing with DDW entirely for 10 min.</li>
		<li>Wipe the scissors and forceps with 70% ethanol.</li>
		<li>Place the mouse in a plastic container with a mesh bottom and with the 2 mL isoflurane-soaked gauze underneath the mesh to avoid the direct contact.</li>
		<li>Asses the depth of anesthesia by pedal reflex (firm toe pinch).</li>
		<li>Remove the mouse when it has reached the desired depth of anesthesia. 
		NOTE: The induction time of anesthesia should not be more than 1 min.</li>
		<li>Construct a nose cone using a 15 mL conical tube with 1 mL isoflurane-soaked pad pushed to the end of the tube.</li>
		<li>Control the depth of anesthesia by moving the nose cone closer or away from the mouse nostrils.</li>
		<li>Place the mouse on the working platform with the ventral side facing up.</li>
		<li>Spray 70% ethanol over the abdominal area of the mouse and then open the abdominal cavity by making a large transverse incision of the dermis and peritoneum by using sharp scissors and toothed forceps.</li>
		<li>Then, make a vertical cut of the abdominal layers until all abdominal organs are completely exposed by using sharp scissors and toothed forceps.</li>
		<li>Move small and large intestines toward the left side of the mouse with autoclaved cotton tip until liver, portal vein, and inferior vena cava (IVC) are clearly exposed.</li>
		<li>Insert a 24 G catheter into IVC just at the bifurcation with right renal vein carefully without shaking hands to avoid the injury of IVC and bleeding. 
		NOTE: If bleeding occurs while you are inserting the catheter into IVC, then you should try to insert the new catheter into a portal vein.</li>
		<li>Remove the needle of the catheter and control the position of the cannula to avoid getting out of IVC, then connect 24 G cannula with perfusion tube by using the connector. 
		NOTE: Avoid the presence of air bubbles before starting the perfusion.</li>
		<li>Start the perfusion of the liver with warm HBSS (-) at a flow rate of 4 mL/min and cut off the splenic vein quickly to drain out the internal blood by using scissors. 
		NOTE: If the cannulation is successful, the liver will begin to blanch due to the backflow distribution of the buffer from IVC to hepatic veins. The portal vein is formed by the union of splenic and superior mesenteric veins; therefore, it is quite safe to cut the splenic vein since it is big and distal from the liver.</li>
		<li>Continue perfusion with 35 mL of warm HBSS (-). 
		NOTE: The liver will become pale in color if the perfusion is adequate, and the mouse still alive under anesthesia at this stage.</li>
	</ol>
	</li>
	<li>Digestion and extraction
	<ol>
		<li>Perfuse the mouse liver with warm 35 mL of HBSS (+) including Collagenase Type X at a flow rate of 4 mL/min.</li>
		<li>Every few minutes, clamp the splenic vein by using forceps for ~10 s to allow the entire volume of HBSS (+) to diffuse into all anatomical parts of the liver. 
		NOTE: Liver begins to swell after clamping the splenic vein due to buffer congestion.</li>
		<li>Pour 5 mL of prepared warm HBSS (+) with Collagenase Type X into 100 mm Petri dish at the end of the perfusion process.</li>
		<li>Cut off the perfused liver from the rest of the body before stopping the pump to avoid the backflow of blood into the liver, remove the gallbladder by forceps, and then wipe the liver gently with a paper towel to remove the blood.</li>
		<li>Transfer the liver to the Petri dish which contains 5 mL of prepared warm HBSS (+) and remove the liver capsule gently using straight-tipped forceps. 
		NOTE: The liver capsule is a thin layer of connective tissue that surrounding the liver.</li>
		<li>Disperse the parenchymal tissue carefully by using the forceps. Add 15 mL cold DMEM to the Petri dish. 
		NOTE: The medium will turn cloudy due to parenchymal cells if the liver is well digested.</li>
		<li>Shake the torn liver gently to release the residual parenchymal cells into the medium and add another 15 mL cold DMEM to the Petri dish to get the rest of the cells.</li>
		<li>Transfer DMEM with the dissolved liver through 100 &#956;m cell strainer into a 50 mL conical tube and keep in ice.</li>
	</ol>
	</li>
	<li>Purification
	<ol>
		<li>Centrifuge the cell suspension at 60 x g for 2 min at 4 &#176;C, carefully remove the supernatant by vacuum aspiration and resuspend the pellet in 10 mL DMEM.</li>
		<li>Add resuspended pellet as a thin layer on the top of 40% density gradient buffer, and centrifuge at 800 x g for 10 min without brake at 4 &#176;C.</li>
		<li>Carefully remove supernatant including the upper layer (DMEM) and the middle layer (dead or non-parenchymal cells), but not the lower layer (Pellet: primary parenchymal hepatocytes).</li>
		<li>Resuspend the primary cells with 2&#8211;3 mL William&#8217;s medium E, and transfer to the new 50 mL tube to avoid contamination of dead cells on the wall of the tube.</li>
		<li>Add 7-8 mL William&#8217;s medium E, mix gently, and centrifuge at 800 x g for 1 min at 4 &#176;C.</li>
		<li>Carefully remove supernatant by vacuum aspiration and then resuspend pellet again with 10, 20 or 30 mL of William&#8217;s medium E (according to the size of the pellet).</li>
		<li>Count the cells by using an automated cell counter.</li>
		<li>Seed with William&#8217;s medium E, keep the plate in an incubator at 37 &#176;C for 2-3 h, then replace the medium with10% FBS DMEM medium with Anti-Anti to remove the dead or unattached cells and for overnight incubation at 37 &#176;C.</li>
		<li>Next day, primary mouse hepatocytes are ready for experimental use.</li>
	</ol>
	</li>
</ol>
2. Chemo-selective Ligation Reaction Assay for the Detection of Nascent Protein Synthesis
<ol>
	<li>Metabolic labeling
	<ol>
		<li>(Pre-treatment) Wash primary hepatocytes with 37 &#176;C warm PBS twice and add the methionine-free DMEM medium for 30 min to deplete the cytoplasmic methionine reserves. 
		NOTE: L-Azidohomoalanine, is an amino acid and analog to methionine, contains an azido moiety that can be incorporated into proteins during biosynthesis of cellular proteins so that depletion of cytoplasmic methionine will enhance the feeding and incorporation of L-Azidohomoalanine into newly synthesized proteins of cultured primary hepatocytes.</li>
		<li>(Treatment) Wash primary hepatocytes with 37 &#176;C warm PBS twice and add the methionine-free DMEM medium with 25 &#956;M AHA (L-Azidohomoalanine) for 4&#8211;6 h.</li>
		<li>Add any specific chemical (i.e. 10 &#956;M rotenone) in the medium for 4&#8211;6 h in presence of AHA in order to investigate its effect on nascent protein expression levels.</li>
		<li>After incubation, remove the medium by vacuum aspiration and wash primary hepatocytes with cold PBS three times.</li>
		<li>Add 200 &#956;L lysis buffer to the plate and incubate for 15-30 min on ice, then tilt the plate and take the whole cell lysate into 1.7 mL tube. 
		NOTE: You should observe that cell lysate is very sticky during the cell harvesting.</li>
		<li>Sonicate the cell lysate on ice for 5 s three times by using a probe sonicator to solubilize the proteins and disperse the DNA.</li>
		<li>Vortex the cell lysate for 5 min, centrifuge the cell lysate at 21,130 x g for 10 min at 4 oC and then transfer the clear supernatant into a new tube. Measure the protein concentration twice. 
		NOTE: At this stage, protein samples can be stored at -20 &#176;C for two weeks if they are not immediately used.</li>
	</ol>
	</li>
	<li>Labeling the Azide-modified nascent protein
	<ol>
		<li>Prepare the components (A+B) by adding 60 &#956;L of component (A) to component (B). 
		NOTE: The solution turns to bright pink in color after adding component (A) to (B).</li>
		<li>Prepare the buffers (D) and (E) according to the protocol.</li>
		<li>Take 20&#8211;40 &#956;g cell lysate and add 50 &#956;L 2x reaction buffer (component A+B), then adjust the volume to 80 &#956;L by adding DDW and vortex for 5 s.</li>
		<li>Add 5 &#956;L CuSO4 (component C), and vortex for 5 s.</li>
		<li>Add 5 &#956;L reaction buffer additive 1 (freshly prepared component D), then vortex for 5 s and wait for 3 min.</li>
		<li>Add 10 &#956;L reconstituted reaction buffer additive 2 (component E), then vortex for 5 s and rotate samples for 20 min at 4 &#176;C by using a multi-rotator machine. 
		NOTE: The solution turns to bright orange in color after adding component (E). The samples should be protected from light during the rotation.</li>
		<li>Add 300 &#956;L methanol and vortex for 5 s, add 75 &#956;L chloroform and vortex for 5 s, then add 200 &#956;L DDW and vortex for 5 s.</li>
		<li>Centrifuge the samples at 21,130 x g and 4 &#176;C for 5 min, then discard the aqueous supernatant and keep the pellet.</li>
		<li>Add 250 &#956;L methanol to the tube, vortex, and spin again for 5 min at 21,130 x g.</li>
		<li>Discard the supernatant, then cover the samples with the lint-free tissue and allow the pellets to air-dry for 15 min at room temperature.</li>
	</ol>
	</li>
	<li>PAGE analysis and western blotting
	<ol>
		<li>Solubilize the pellet in 20 &#956;L of loading buffer without &#946;-mercaptoethanol, vortex for 10 min, heat at 70 &#176;C by using heat block for 10 min. Then load the samples into 10% SDS gel (1.5 mm) for Tetramethylrhodamine (TAMRA) detection.</li>
		<li>Detect AHA signal using a variable mode laser scanner for precise quantitation of nascent proteins.</li>
		<li>Wash the Gel with DDW for 15 min.</li>
		<li>Then, stain the 10% SDS gel with a fast and sensitive coomassie-dye reagent for 15&#8211;60 min to detect total proteins as a control.</li>
		<li>De-stain the Gel with DDW for 1&#8211;2 h and acquire the image by using a UV fluorescence imager.</li>
	</ol>
	</li>
</ol>

The authors indicate they have no conflicts of interest.

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 physiology and metabolism in culture, despite the challenges of culturing and maintaining of these cells. Herein, our study details the successful isolation of primary mouse hepatocytes by conducting a two-step collagenase method in a relatively convenient and efficient manner. Through these procedures, the majority of isolated primary hepatocytes within the culture retain their metabolic functionality and morphological stability and can be used for investigating hepatic functions including protein metabolism, gluconeogenesis, and mitochondrial respiration.
Isolated primary hepatocytes are a powerful experimental tool to examine molecular mechanisms of hepatic energy metabolism. Rotenone interferes with the electron transport chain complex I activity in mitochondria and blocks oxidative phosphorylation with the limited synthesis of ATP53, as evidenced by the activation of AMPK (Figure 4). The AMPK activation triggers the cellular adaptive responses that suppress the anabolic mechanism to compensate the rotenone-induced energy-deficient stress. Under this condition, we successfully detected the drastic reduction of nascent protein synthesis levels in primary hepatocytes using a non-radioactive, chemo-selective ligation reaction with a TAMRA protein detection method (Figure 5). These results confirm the importance of energy supply from the mitochondria for appropriate protein synthesis in primary hepatocytes. These techniques are useful to provide the information regarding a drug action or toxicity within therapeutically relevant concentrations in the context of hepatic protein synthesis of healthy and diseased conditions. In addition, these techniques are applicable for examining the role of specific pathways modulating protein and energy metabolism by analyzing primary hepatocytes isolated from genetically manipulated mice.
In general, low yield and cell viability are the major drawbacks of the use of primary hepatocytes. These issues are due to several reasons such as initial cannulation, insufficient washing with HBSS, inappropriate concentration of collagenase, or long-perfusion period. Our current protocol optimizes these multiple steps and leads to gaining a higher yield and viable cells around 20 million/liver/adult mice. Especially, there are two critical steps: one is to adjust the temperature and the flow rate of digested buffers with proper collagenase concentration to digest the hepatic parenchyma efficiently in 15&#8211;20 min from cannula insertion until liver excision, and another is to process these collagenases treated samples quickly as described in order to keep cells in healthy condition. While isolated hepatocytes-based studies have been conducted within 48 h time frame in many published data, primary hepatocytes isolated with our procedures endure the longer-term culture (Figure 2). With these cells, we show the robust labeling of hepatic nascent protein synthesis through the chemo-selective ligation reaction assay, although the concentration and incubation time with AHA in the protocol may have to be re-optimized in different biological conditions of cells (e.g. physiological vs. pathological, wild-type vs. transgenic or knockout/down cells).
In summary, we have established procedures to analyze primary mouse hepatocytes for nascent protein synthesis in a non-radioactive manner. These cells respond well to mitochondrial stress and exhibit suppression of protein synthesis with activation of the AMPK pathway. Thus, these isolated hepatocytes are valuable models for investigating the pathophysiology of hepatic protein and energy metabolism ex vivo.

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&#8211;30% of total energy consumption5,6. In addition, proteins function as not only passive or building blocks of the liver, but also active signal-mediating factors intracellularly or extracellularly to regulate the systemic metabolism7. For instance, reduced levels of serum albumin, which is synthesized and secreted by the liver and the most abundant protein in the plasma8, increases the risk of type 2 diabetes development9,10,11, whereas a higher concentration of serum albumin is protective against developing metabolic syndrome12. Furthermore, disturbed or disruption of secretory or membrane-bound hepatic proteins, which modulate cholesterol homeostasis, including lipoproteins, LDLR, and LRP1, can lead to the development of insulin resistance, hyperlipidemia, or atherosclerosis13. Therefore, the identification of molecular pathophysiological mechanisms that are involved in protein metabolism disturbance in the liver and its associated metabolic complications might be useful for discovering novel pharmacological approaches to retard onset or treat metabolic diseases such as insulin resistance, diabetes, and non-alcoholic fatty liver.
Protein synthesis is tightly linked to the cellular energy status (e.g., the formation of one peptide bond during the elongation step of protein synthesis requires 4 phosphodiester bonds14) and is regulated by molecular pathways that sense inter- and intra-cellular nutrient availability15,16. AMP-activated protein kinase (AMPK) is one of the intracellular energy sensors that maintain energy homeostasis17. Once AMPK is activated when cellular energy levels become lower, AMPK and its targeted substrates function to stimulate catabolic pathways and inhibit anabolic processes including protein synthesis18,19. The regulation of protein synthesis is mediated by phosphorylation of multiple translation factors and ribosomal proteins20. Of note, the mammalian target of rapamycin complex 1 (mTORC1), a major driver of protein synthesis, is one of the main targets of AMPK21. Activation of the mTORC1 pathway enhances the cell growth and proliferation by stimulating protein translation and autophagy20,21. Therefore, it is logical that activation of AMPK can inhibit mTORC1-mediated protein synthesis22. Indeed, activation of AMPK counteracts and directly phosphorylates mTORC1 on threonine residue 2446 (Thr2446) leading to its inactivation23 and suppression of protein biosynthesis24. Moreover, AMPK can indirectly inhibit mTORC1 function by phosphorylation and activation of tuberous sclerosis complex 2 (TSC2)25 which is the upstream regulator of mTORC1 signaling cascade. In short, dysregulation of these pathways in the liver is often linked to the development of metabolic diseases and therefore there is a critical need to establish effective experimental tools to investigate the role of these pathways in the regulation of energy and protein metabolism in hepatocytes.
There is a stronger similarity between the functional properties of isolated primary hepatocytes and in vivo hepatocytes than with in vitro liver-derived cell lines26,27,28. It has been shown that primary human hepatocytes share 77% similarity with that of liver biopsies, whereas HepG2 cells, which are well-differentiated hepatic cancerous cells and widely used to investigate hepatic functions, display less than 48% in the context of gene expression profiles29. Therefore, utilization of primary hepatocytes, rather than immortalized culture cells, is of vital importance in investigating hepatic function and physiology, and several protocols are available for the isolation and culture of primary hepatocytes especially from rats30,31. While the rat hepatocytes are useful with a relatively higher yield of cells, mouse hepatocytes have a greater potential in many scientific aspects because of the wide availability of genetically modulated mice. However, there are several technical challenges in isolating healthy and abundant primary hepatocytes from mice for cellular- and molecular-based assessments: first, cannula insertion to perfuse the liver with buffer reagents is very difficult to handle because of the small and thin mouse portal vein or inferior vena cava; second, a longer manipulation time of cells during the isolation can cause reduction in cell quantity and quality; third, non-enzymatic mechanical separation methods can result in severe damage and produce a low yield of viable isolated primary hepatocytes32,33. In the 1980s, collagenase perfusion technique was introduced for isolating hepatocytes from the livers of animals34. This method is based on collagenase perfusion of the liver35,36,37, infusion of the liver with calcium chelator solution38,39, enzymatic digestion and mechanical dissociation of the hepatic parenchyma35. In the first step, a mouse liver is perfused with a calcium [Ca2+] free buffer containing a [Ca2+] chelator (ethylenediamine tetraacetic acid, EDTA). In the second step, the mouse liver is perfused with a collagenase-containing buffer to hydrolyze the cellular-extracellular matrix interactions. Unlike the buffer used in step one, the presence of [Ca2+] ions in the buffer of the second step is required for effective collagenase activity, after which the digested liver has to be further gently and mechanically separated using forceps between the hepatic capsule and parenchymatous tissue. Finally, connective tissue is removed by filtering, and subsequent centrifugation separates viable hepatocytes from both non-parenchymal cells and non-living hepatocyte with the use of density gradient buffer40,41,42. In the present study, we show a modified two-step collagenase perfusion technique to isolate primary hepatocytes from a mouse liver for the analysis of protein synthesis.
The radiolabeling of proteins is widely used to quantify the expression levels, turnover rates, and determine the biological distribution of proteins43 due to the high sensitivity of detection of radioactivity44. However, the use of radioactive isotopes requires highly controlled research circumstances and procedures45. Alternative non-radioactive methods have been developed and have increasingly gained in popularity. The chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method is one of them and is based on the chemo-selective reaction between an azide and alkyne groups46, which can be utilized to analyze cellular events such as detecting nascent protein synthesis and subclasses of glycoproteins modified with an azide group. For nascent protein synthesis, L-Azidohomoalanine (L-AHA, an azide-modified amino acid) can be metabolically incorporated into proteins and detected by using the TAMRA protein detection method47. By using this assay in primary mouse hepatocytes, we show that the nascent protein synthesis rate is tightly linked to the availability of ATP from mitochondrial and AMPK activation (Figure 1).
In summary, utilization of primary mouse hepatocytes is crucial to investigating the protein and energy metabolism and quantifying nascent protein synthesis is valuable for gaining insights into the physiological role of pathways relevant to the development and cure of hepatocyte-related diseases.

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

isolation of primary mouse hepatocytes for nascent protein synthesis analysis by non-radioactive l-azidohomoalanine labeling method

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.

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 isolated primary mouse hepatocytes (PMHs), mouse embryonic fibroblasts (MEFs), a mouse hepatoma cell line (Hepa 1-6), and mouse livers. The albumin protein expression was detected in primary mouse hepatocytes and mouse livers but was not detectable in either MEFs or Hepa 1-6 cells (Figure 3).
To determine whether the effects of rotenone on hepatic AMP-activated protein kinase (AMPK) activation is cell-autonomous, we compared the time-dependent response of primary hepatocytes isolated from a liver of wild-type mouse. Primary hepatocytes were treated without or with 10 &#956;M rotenone for 0, 4 or 6 h. In these cells, rotenone treatment robustly induced AMPK activation, as detected by increased phosphorylation levels on AMPK-T172 similar to those seen in vivo48 (Figure 4).
To directly measure the effects of rotenone treatment on nascent protein synthesis in primary hepatocytes, the incorporation of non-radioactive AHA into protein over 5 h was assessed by performing the chemo-selective ligation reaction assay. Treatment with 10 &#956;M rotenone for 5 h had profound inhibitory effects on nascent protein synthesis in treated primary hepatocytes when compared to untreated primary hepatocytes (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58323/58323fig1v2.jpg" /> 
Figure 1: An illustration shows the steps of primary mouse hepatocytes isolation, western blot analysis, and chemo-selective ligation reaction procedures.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58323/58323fig2.jpg" /> 
Figure 2: Phase contrast morphological images of primary mouse hepatocytes. Representative pictures of isolated primary hepatocytes were acquired at 2, 12, 24, and 72 h after plating. Scale bars = 100 &#956;m.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58323/58323fig3.jpg" /> 
Figure 3: Western blot analysis for albumin protein expression in primary mouse hepatocytes. Western blot analysis was conducted with the loading of 10 &#956;g protein/lane from primary mouse hepatocytes (PMHs), mouse embryonic fibroblasts (MEFs), Hepa 1-6 cells, and mouse livers. One-way ANOVA showed a significant albumin protein expression in PMHs compared to MEF or Hepa 1-6 cells. Values are means &#177; SEM of group size (n = 3). *P, #P &#60;0.001 vs. Hepa 1-6 or MEFs, respectively. $P &#60;0.001 vs. liver lysate.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58323/58323fig4.jpg" /> 
Figure 4: Induction of phosphorylation of AMPK in rotenone-treated primary mouse hepatocytes. Primary hepatocytes from a wild-type mouse liver were treated with 10 &#956;M rotenone for 0, 4 or 6 h. Phosphorylation levels of AMPK were detected by western blotting and &#946;-actin was used as a loading control (10 &#956;g protein was loaded/lane). Repeated measurements 2-way ANOVA showed that treatment caused a significant increase in protein expression of phospho-AMPK in comparison to untreated primary hepatocytes. Values are means &#177; SEM of group size (n = 3). *P &#60;0.001 vs. Untreated hepatocytes.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58323/58323fig5.jpg" /> 
Figure 5: Reduction of hepatic nascent protein expression after rotenone treatment. Primary hepatocytes from a wild-type mouse liver were treated with 10 &#956;M rotenone for 5 h, followed by a nascent protein synthesis assay with non-radioactive AHA method. Protein synthesis levels were assessed by a laser scanner detecting the TAMRA fluorescence. Total protein loading levels for this assay were detected by the coomassie-dye reagent.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Isolation of primary mouse hepatocytes</td>
			<td>Chemoselective ligation reaction assay for detection of nascent protein synthesis</td>
		</tr>
		<tr>
			<td>1.&#160;HBSS (-) Buffer 500 ml:</td>
			<td>1. Protein analysis detection Kit, TAMRA alkyne&#160;</td>
		</tr>
		<tr>
			<td>1.1. HEPES: 1.1915 g</td>
			<td>1.1. Component A (TAMRA alkyne)</td>
		</tr>
		<tr>
			<td>1.2. D-glucose: 0.9 g&#160;</td>
			<td>1.2. Component B (reaction buffer)</td>
		</tr>
		<tr>
			<td>1.3. EGTA: 0.095 g&#160;</td>
			<td>1.3. Component C (CuSO4)</td>
		</tr>
		<tr>
			<td>1.4. Anti-Anti: 5 ml</td>
			<td>1.4. Component D (reaction buffer additive 1)</td>
		</tr>
		<tr>
			<td>1.5. HBSS (10 X): 50 ml</td>
			<td>1.5. Component E (reaction buffer additive 2)</td>
		</tr>
		<tr>
			<td>1.6.&#160; Add distilled deionized water (DDW) up to 500 ml</td>
			<td>2. AHA: L-azidohomoalanine&#160;</td>
		</tr>
		<tr>
			<td>(Note: Adjust pH to 7.4, Filter, and Store at 4 oC)</td>
			<td>3. Methionine-free DMEM medium</td>
		</tr>
		<tr>
			<td>2. HBSS (+) Buffer 500 ml:</td>
			<td>3.1. DMEM: 500 mL&#160;&#160;</td>
		</tr>
		<tr>
			<td>2.1. HEPES: 1.1915 g&#160;</td>
			<td>3.2. FBS: 56 mL&#160;&#160;</td>
		</tr>
		<tr>
			<td>2.2. CaCl2.2H2O: 0.3675 g&#160;</td>
			<td>3.3. Anti-Anti: 5.6 mL&#160;</td>
		</tr>
		<tr>
			<td>2.3. Anti-Anti: 5 ml&#160;</td>
			<td>3.4. L-Cystine Dihydrochloride&#160;</td>
		</tr>
		<tr>
			<td>2.4. HBSS (10 X): 50 ml&#160;</td>
			<td>3.5.L-alanyl-L-glutamine dipeptide: 5.6 mL&#160;</td>
		</tr>
		<tr>
			<td>2.5. Add DDW up to 500 ml</td>
			<td>(Note: Store at 4 oC)</td>
		</tr>
		<tr>
			<td>(Note: Adjust pH to 7.4, Filter, and Store at 4 oC)</td>
			<td>4. Warm PBS</td>
		</tr>
		<tr>
			<td>3. 40% density gradient buffer 450 ml:</td>
			<td>5. Lysis buffer</td>
		</tr>
		<tr>
			<td>3.1. density gradient buffer: 180 ml&#160;</td>
			<td>5.1. 50 mM Tris-HCl</td>
		</tr>
		<tr>
			<td>3.2. HBSS (10 X): 45 ml&#160;&#160;</td>
			<td>5.2. 1% SDS</td>
		</tr>
		<tr>
			<td>3.3. DDW: 225 ml</td>
			<td>5.3. Protease inhibitor</td>
		</tr>
		<tr>
			<td>(Note: Filter and Store at 4 oC)</td>
			<td>5.4. Phosphatase inhibitor</td>
		</tr>
		<tr>
			<td>4.&#160;DMEM:</td>
			<td></td>
		</tr>
		<tr>
			<td>4.1. DMEM: 500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>4.2. FBS: 56 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>4.3. Anti-Anti: 5.6 ml&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>(Note: Store at 4 oC)</td>
			<td></td>
		</tr>
		<tr>
			<td>5.Williams&#8217; Medium E:</td>
			<td></td>
		</tr>
		<tr>
			<td>5.1. Williams&#8217; Medium E: 500 ml&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>5.2. FBS: 30 ml&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>5.3. Anti-Anti: 5.4 ml&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>5.4. L-alanyl-L-glutamine dipeptide: 5.4 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>(Note: Store at 4 oC)</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1

Watch this Scientific Journal Video about Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method at JoVE.com

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

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 ...

isolation-primary-mouse-hepatocytes-for-nascent-protein-synthesis

Nanyang Technological University

Research

JoVE Journal

Medicine

18.7K Views.  University of Cincinnati. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in verschillende gebieden van metabole ziekte onderzoek over moleculaire veranderingen van leverenergie en eiwit biosynthese in obesitas, niet-alcoholische vette lever, en type twee diabetes. De belangrijkste voordelen van deze techniek zijn dat het de isolatie van functionele primaire muis hepatocyten en detectie leververblijvende eiwitten via een niet-radioactieve etikettering substraat vergemakkelijkt. Visuele demonstratie...

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

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Biochemistry

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 &#215; 106 cells per liver and cell viability of &#62;85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of &#62;35% in mouse hepatocytes.
To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.

This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

Bioengineering

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol describes in vivo labeling and subsequent purification of newly synthesized proteins using an engineered mouse line to direct protein labeling to specific cellular populations. It is an inducible line by Cre recombinase expression of L274G-Methionine tRNA synthetase (MetRS*), enabling azidonorleucine (ANL) incorporation to the proteins, which otherwise will not occur. Using the method described here, it is possible to purify cell-type-specific proteomes labeled in vivo and detect subtle changes in protein content due to sample complexity reduction.

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

Understanding protein homeostasis in vivo is key to knowing how the cells work in both physiological and disease conditions. The present protocol ...

Neuroscience

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Biology

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

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

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