This work was supported by the NIH (R01HL128714 to C.F.) and (F31DK10236101 to K.S) and the American Heart Association (C.F. and A.G.).

All animal protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Miami Miller School of Medicine.
1. Preparation
<ol>
	<li>Preheat liver perfusion media and liver digest media in a water bath at 37 &#176;C.</li>
	<li>Set up a perfusion pump (30 mL/min). Carefully prefill the tubing system of the pump with liver perfusion media. Avoid any air bubbles in the system and prepare the stereotactic microscope.</li>
	<li>Coat cell culture dishes using collagen type I according to the manufacturer&#39;s protocol.</li>
</ol>
2. Liver Recovery
<ol>
	<li>Anesthetize the donor mouse using oxygen/isoflurane inhalation (isoflurane 4%/2 L/min oxygen).
	<ol>
		<li>Maintain anesthesia by lowering isoflurane to 2-2.5% or by injecting ketamine (100 mg/kg of body weight intraperitoneally) and xylazine (10 mg/kg of body weight intraperitoneally). Continuous isoflurane anesthesia is preferred since ketamine lowers blood pressure and can cause hepatotoxicity.</li>
	</ol>
	</li>
	<li>Place the anesthetized mouse on an absorbent pad. Fix the mouse&#39;s extremities with adhesive tape.</li>
	<li>Shave and disinfect the abdomen of the mouse and perform a ventral laparotomy from the pubis to the cranial border of the liver using surgical scissors with sharp/blunt tips. Incise the abdominal wall to both sides, caudal of the diaphragm.</li>
	<li>Expose the inferior vena cava (IVC) by carefully moving the intestine to the right side. Carefully dissect fat tissue around the IVC using sterile cotton tip swabs and angled tip forceps.</li>
	<li>Incise the suprahepatic diaphragm and open the thoracic cavity by two lateral incisions using fine surgical scissors with sharp/sharp tips. Ligate the thoracic IVC using surgical silk (5/0).</li>
	<li>Cannulate the infrarenal IVC using a shielded i.v. catheter, carefully remove the needle, connect the perfusion pump and start perfusing the liver. If performed properly, the liver should immediately begin to blanch and swell.</li>
	<li>Transect the portal vein allowing suitable drainage of blood and perfusion media using fine surgical scissors.</li>
	<li>Perfuse the liver with approximately 30 mL of liver perfusion media, and then switch to liver digest media (30 mL). Turn off the perfusion pump and remove the cannula once perfusion is complete.</li>
	<li>Gently expose the central region of the liver and locate the connecting tissue linking the liver lobes. Grab the central connecting fibers and carefully dissect all tissues holding the liver in place and gently remove the liver.</li>
	<li>Immediately transfer the liver into a 50 mL polypropylene conical tube containing 15 mL of liver digest media. 
	NOTE: &#160;There is no specific time-line for this step. After perfusion, the liver remains in digest media for the transport to a sterile cell culture hood.&#160;All subsequent steps should be performed in a sterile environment.</li>
</ol>
3. Isolation and Treatment of Cells
<ol>
	<li>Within a cell culture hood, transfer the liver from the 50 mL polypropylene conical tube containing digestion media into a sterile tissue culture dish.</li>
	<li>Using sterile forceps, gently tear the four lobes of the liver. If performed properly, digestion medium should become turbid due to isolating hepatocytes from the liver.</li>
	<li>Using a 25 mL sterile pipette tip, filter the turbid digestion medium through a 70 &#181;m nylon cell strainer into a new 50 mL polypropylene conical tube to remove undigested tissue particles and cell debris.</li>
	<li>Repeat steps 3.2 and 3.3 with sterile phosphate-buffered saline (PBS). This assists with removing any residual hepatocytes from the liver.</li>
	<li>Centrifuge at 50 x g for 2 min at 4 &#176;C. Aspirate the supernatant containing excess cell debris and gently re-suspend cells in 25 mL cold 1x Williams&#39; Medium E.</li>
	<li>Using a 25 mL sterile pipette tip, filter the re-suspension through a new 70 &#181;m nylon cell strainer into a 50 mL polypropylene conical tube to achieve a cleaner suspension.</li>
	<li>Repeat steps 3.5 and 3.6 for three additional washes using cold Williams&#39; Medium E 1x to obtain a clearer cell suspension.</li>
	<li>After the final washing step, aspirate supernatant to remove excess cell debris. Re-suspend and filter cells in 25 mL warm Williams&#39; Media E 1x that contains primary hepatocyte thawing and plating supplements through a new 70 &#181;m nylon cell strainer into a 50 mL polypropylene conical tube.</li>
	<li>Count cells within the 25 mL cell suspension using a hemocytometer. On average expect 15 - 20 million cells per adult liver. Determine cell viability using trypan blue staining.</li>
	<li>Plate cells in 2 mL of Williams&#39; Media E 1x that contains primary hepatocyte thawing and plating supplements on a collagen coated 6-well cell culture cluster (9 x 105 cells per well).</li>
	<li>After 4 h, change media to Williams&#39; Media E 1x that contains primary hepatocyte maintenance supplements.</li>
	<li>After 24 h, serum starve cells with Dulbecco&#39;s modified Eagle medium (DMEM) media 1x for 6 h.</li>
	<li>Treat cells with PBS as vehicle, FGF23 (25 ng/mL), LPS (100 &#181;g/mL) or IL-6 (50 ng/mL) in serum-free medium and incubate for 24 h.</li>
</ol>
4. Isolation of RNA
<ol>
	<li>Prepare RNA using a commercial spin column kit according to the manufacturer&#39;s instructions.</li>
</ol>
5. Generating cDNA from isolated RNA by Reverse Transcription
<ol>
	<li>Add 200 ng of isolated RNA sample to a PCR tube.</li>
	<li>Add 14 &#181;L of endonuclease-free H2O to PCR tube from step 5.1.</li>
	<li>Add 4 &#181;L of reverse transcriptase to PCR tube from step 5.1.</li>
	<li>Re-suspend contents gently to make mixture homogenous; centrifuge gently.</li>
	<li>Place PCR tube containing the mixture into a thermocycler. Run thermocycler for reverse transcription: 1 cycle of 25 &#176;C (5 min), 42 &#176;C (30 min), 85 &#176;C (5 min) and then hold at 4 &#176;C. The final product will be the desired cDNA.</li>
</ol>
6. Analysis of Cells by Quantitative Real-time PCR (qPCR)
<ol>
	<li>Prepare the 96-well plate.
	<ol>
		<li>Place all components of qPCR reaction on ice: 1 &#181;L cDNA, .25 &#181;M forward primer, .25 &#181;M reverse primer, 5 &#181;L SYBR Green, 3.5 &#181;L PCR grade water to a total volume of 10 &#181;L. Using SYBR Green, prepare master mix to run all samples in triplicate.</li>
		<li>Once the master mix is complete, vortex to make homogenous. Aliquot 9 &#181;L of qPCR master mix into each well of a 96-wll plate. After, carefully add 1 &#181;L of cDNA into each well. Total reaction volume will become 10 &#181;L.</li>
		<li>After loading is complete, seal 96-well plate with optical adhesive film and briefly centrifuge 96-well plate (50 x g for 1 min).</li>
	</ol>
	</li>
	<li>Run samples in a Real Time-PCR instrument. Place sealed 96-well plate into Real Time-PCR instrument and run samples as per instrument manufacturer recommendations. Examples of standard and fast cycles are included below.
	<ol>
		<li>For standard cycling parameters, use the following: initial denaturation at 94 &#176;C for 120 s, then 40 cycles of 94 &#176;C for 15 s, 60 &#176;C for 60 s, and then optionally hold at 4 &#176;C.</li>
		<li>For fast cycling parameters: use the following: initial denaturation at 94 &#176;C for 30 s, then 40 cycles of 95 &#176;C for 5 s, 58 &#176;C for 15 s, and then 72 &#176;C for 10 s.</li>
	</ol>
	</li>
	<li>Refer to instrument manual for guidance on how to analyze data.</li>
</ol>
7. Analysis of Cell Supernatants by ELISA
NOTE: All steps are performed according to manufacturer&#39;s protocol.
<ol>
	<li>Sample Preparation
	<ol>
		<li>Dispense 398 &#181;L of diluent into separate tubes. Pipette 2 &#181;L of the cell supernatant sample into the 398 &#181;L diluent tube. This will provide a dilution fold of 200.</li>
		<li>Repeat step 7.1.1 for each sample to be tested.</li>
	</ol>
	</li>
	<li>Procedure
	<ol>
		<li>Pipette 100 &#181;L of 200-fold diluted sample and a standard into the wells of a 96-well plate.</li>
		<li>Incubate the 96-well plate at 25 &#176;C for 45 min on a microplate shaker at 150 rpm.</li>
		<li>Wash the wells 5 times with 1x wash buffer solution. Once the last&#160;wash is completed, remove all residual wash solution with absorbent paper.</li>
		<li>Add 500 &#181;L of HRP-conjugate reagent into each well.</li>
		<li>Repeat steps 7.2.2 - 7.2.3.</li>
		<li>Pipette 100 &#181;L of tetramethylbenzidine (TMB) reagent into each well of 96-well plate.</li>
		<li>Gently mix 96-well plate at 25 &#176;C for 20 min on a microplate shaker at 150 rpm. Stop the reaction by directly adding 100 &#181;L of stop solution to each well and mix gently. Once the stop solution is added the color should turn yellow. This informs that the stop solution has been correctly mixed.</li>
		<li>Using a microtiter plate reader, determine the optical density at 450 nm within the first 5 min.</li>
	</ol>
	</li>
	<li>Calculation of Results
	<ol>
		<li>Calculate a table of average absorbance values (A450) for each sample and each standard.</li>
		<li>Assemble a standard curve by plotting the A450 mean of each standard against its standard concentration in ng/mL on a linear graph. Allow the x-axis to represent the concentration and the y-axis to represent A450 values.</li>
		<li>Utilizing the mean A450 from each diluted sample, determine the concentration of the desired protein in ng/mL from the standard curve.</li>
		<li>Multiply the sample concentration by the dilution factor. This will allow for determining the actual concentration of the desired protein in the cell supernatant sample.</li>
		<li>Plot the constructed table from samples into a line graph. If OD450 values fall outside of the standard curve, samples should be diluted appropriately and re-tested.</li>
		<li>Normalize results to 500,000 cells.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Silverstein, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+chronic+kidney+disease:+role+in+the+progression+of+renal+and+cardiovascular+disease.">Inflammation in chronic kidney disease: role in the progression of renal and cardiovascular disease.</a> Pediatr Nephro. 24 (8), 1445-1452 (2008).</li><li>Isakova, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+23+is+elevated+before+parathyroid+hormone+and+phosphate+in+chronic+kidney+disease.">Fibroblast growth factor 23 is elevated before parathyroid hormone and phosphate in chronic kidney disease.</a> Kidney Int. 79 (12), 1370-1378 (2011).</li><li>Faul, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF23+induces+left+ventricular+hypertrophy.">FGF23 induces left ventricular hypertrophy.</a> J Clin Invest. 121 (11), 4393-4408 (2011).</li><li>Guti&#233;rrez, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+23+and+mortality+among+patients+undergoing+hemodialysis.">Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis.</a> N England J Med. 359 (6), 584-592 (2008).</li><li>Munoz Mendoza, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+23+and+Inflammation+in+CKD.">Fibroblast growth factor 23 and Inflammation in CKD.</a> Clin J Am Soc Nephrol. 7 (7), 1155-1162 (2012).</li><li>Hanks, L. J., Casazza, K., Judd, S. E., Jenny, N. S., Guti&#233;rrez, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+of+Fibroblast+Growth+Factor-23+with+Markers+of+Inflammation,+Insulin+Resistance+and+Obesity+in+Adults.">Associations of Fibroblast Growth Factor-23 with Markers of Inflammation, Insulin Resistance and Obesity in Adults.</a> PLoS ONE. 10 (3), e0122885 (2015).</li><li>Singh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+23+directly+targets+hepatocytes+to+promote+inflammation+in+chronic+kidney+disease.">Fibroblast growth factor 23 directly targets hepatocytes to promote inflammation in chronic kidney disease.</a> Kidney Int. , 1-12 (2016).</li><li>Howard, R. B., Christensen, A. K., Gibbs, F. A., Pesch, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enzymatic+preparation+of+isolated+intact+parenchymal+cells+from+rat+liver.">The enzymatic preparation of isolated intact parenchymal cells from rat liver.</a> J Cell Biol. 35 (3), 675-684 (1967).</li><li>Moshage, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+interleukin-1,+interleukin-6+and+its+interrelationship+on+the+synthesis+of+serum+amyloid+A+and+C-reactive+protein+in+primary+cultures+of+adult+human+hepatocytes.">The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes.</a> Bioch Biophysi Res Commun. 155 (1), 112-117 (1988).</li><li>Soldatow, V. Y., LeCluyse, E. L., Griffith, L. G., Rusyn, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+for+liver+toxicity+testing.">In vitro models for liver toxicity testing.</a> Toxicol. Res. 2 (1), 23-39 (2013).</li><li>Lee, S. M. L., Schelcher, C., Demmel, M., Hauner, M., Thasler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Human+Hepatocytes+by+a+Two-step+Collagenase+Perfusion+Procedure.">Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure.</a> J Vis Exp. (79), (2013).</li><li>Kegel, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+Isolation+of+Primary+Human+Hepatocytes+and+Corresponding+Major+Populations+of+Non-parenchymal+Liver+Cells.">Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells.</a> J Vis Exp. (109), (2016).</li><li>Moshage, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+and+the+hepatic+acute+phase+response.">Cytokines and the hepatic acute phase response.</a> J Pathol. 181 (3), 257-266 (1997).</li><li>Klaunig, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+liver+cell+culture.+I.+Hepatocyte+isolation.">Mouse liver cell culture. I. Hepatocyte isolation.</a> In vitro. 17 (10), 913-925 (1981).</li><li>Lu, Y. C., Yeh, W. C., Ohashi, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS/TLR4+signal+transduction+pathway.">LPS/TLR4 signal transduction pathway.</a> Cytokine. 42 (2), 145-151 (2008).</li><li>Schmidt-Arras, D., Rose-John, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-6+pathway+in+the+liver:+From+physiopathology+to+therapy.">IL-6 pathway in the liver: From physiopathology to therapy.</a> J Hepatol. 64 (6), 1403-1415 (2016).</li><li>Grabner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Cardiac+Fibroblast+Growth+Factor+Receptor+4+Causes+Left+Ventricular+Hypertrophy.">Activation of Cardiac Fibroblast Growth Factor Receptor 4 Causes Left Ventricular Hypertrophy.</a> Cell Metab. 22 (6), 1020-1032 (2015).</li><li>Shulman, M., Nahmias, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17,+Long-Term+Culture+and+Coculture+of+Primary+Rat+and+Human+Hepatocytes.">Chapter 17, Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes.</a> Methods Mol Biol. 945, 287-302 (2012).</li></ol>

The authors have no conflict of interests to declare.

Isolating primary hepatocytes from mice is a fast, inexpensive and reliable tool to study inflammatory responses ex vivo. If performed correctly, results can be easily generated and reproduced in a timely and cost-efficient manner. The following points should be carefully assessed in order to ensure a successful isolation.
The surgical incision and the cannulation of the IVC should be performed under general anesthesia and not after euthanasia. A young, inexperienced investigator will...

Chronic kidney disease (CKD) can be defined as a state of acute and chronic inflammation1. In patients with CKD, serum levels of the phosphaturic hormone fibroblast growth factor 23 (FGF23) progressively rise in order to maintain serum phosphate homeostasis2. Increased serum FGF23 levels are independently associated with cardiovascular morbidity and mortality among patients who are beginning hemodialysis treatment3,4. Furthermore, several clinical studies have shown a strong correlation between elevated FGF23 levels and serum levels of C-reactive protein (CRP), Interleukin-6 (IL-6) and Tumor Necrosis Factor &#945; (TNF&#945;)5,6. Moreover, in an experimental study, we have recently demonstrated that FGF23 can directly target hepatocytes and cause an inflammatory response by increasing CRP and IL-6 production in the liver7. Hence, FGF23 might act as a circulating factor that contributes to systemic inflammation in CKD.
In the early 70&#39;s, primary hepatocytes were isolated and studied for the first time8. Since then primary cultured hepatic cells have been extensively used to examine metabolic processing, hormonal function, drug metabolism and toxicity as well as immunity and inflammatory responses9,10. Previous protocols have mainly described the enzymatic isolation of primary hepatocytes from human liver tissue11,12. While an excellent model, this leaves out the ability to study how genetic manipulation affects complex hepatic signaling mechanisms as well as functional consequences upon different types of stimuli. In the following, we describe the isolation of murine primary hepatocytes. Notably, the effect of several mediators of the hepatic acute-phase response, such as lipopolysaccharide (LPS), IL-6 and FGF23 can be analyzed in an easy, fast and reproducible manner13.
Herein, we present a protocol for the enzymatic isolation of hepatocytes from adult mice, and we demonstrate that established inducers of inflammation, such as LPS and IL-6, as well as novel inflammatory mediators such as FGF23, can directly stimulate expression and secretion of inflammatory cytokines, such as CRP and IL-6 in cultured hepatocytes.

<table border="0" cellpadding="0" cellspacing="0" width="924">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Consumables</td>
			<td style="width:283px;"></td>
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			<td style="width:103px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer 70 &#956;m Nylon cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
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			<td>BD</td>
			<td>381412</td>
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			<td height="21" style="height:21px;">50 mL Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352098</td>
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			<td height="21" style="height:21px;">100 6-inch Cotton Tipped Applicators&#160;</td>
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			<td>Becton Dickinson</td>
			<td>329420</td>
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			<td height="21" style="height:21px;">Tissue Culture Dish 100 x 20 mm Style</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
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			<td height="21" style="height:21px;">6 well Cell Culture Cluster</td>
			<td>Costar</td>
			<td>3516</td>
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			<td height="21" style="height:21px;">5/0 Black Braided Surgical Silk (100 yards)</td>
			<td>LOOK</td>
			<td>SP115</td>
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			<td height="21" style="height:21px;">Name</td>
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			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
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			<td height="21" style="height:21px;">Minipuls 3 Perfusion Pump</td>
			<td>Gilson</td>
			<td>F155007</td>
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			<td height="21" style="height:21px;">Hemacytometer Kits, Propper</td>
			<td>VWR</td>
			<td>48300-474</td>
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			<td>48300-470</td>
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			<td height="21" style="height:21px;">Surgical Scissers - Sharp/Blunt</td>
			<td>F.S.T.</td>
			<td>14001-12</td>
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			<td height="22" style="height:23px;">Iris Scissors-ToughCut Straight</td>
			<td>F.S.T.</td>
			<td>14058-11</td>
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			<td height="22" style="height:23px;">Dumont SS-45 Forceps</td>
			<td>F.S.T.</td>
			<td>11203-25</td>
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			<td height="22" style="height:23px;">Student Tissue Forceps</td>
			<td>F.S.T.</td>
			<td>991121-12</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
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			<td height="21" style="height:21px;">Reagents</td>
			<td></td>
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			<td height="21" style="height:21px;">Acetic acid solution, 2.0 N</td>
			<td>Sigma</td>
			<td>&#160;&#160;&#160; A8976-100ML</td>
			<td></td>
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			<td height="21" style="height:21px;">Isoflurane, USP 250 mL</td>
			<td>Piramal Healthcare</td>
			<td>&#160;&#160; 66794-013-25</td>
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			<td>VEDCO</td>
			<td>&#160;&#160;&#160; 50989-161-06</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Xylazine 100 mg/mL</td>
			<td>AnaSed Injection</td>
			<td>139-236</td>
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		</tr>
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			<td>gibco</td>
			<td>12551-032</td>
			<td></td>
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			<td>gibco</td>
			<td>17701-038</td>
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			<td>Life Technologies</td>
			<td>17703034</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary Hepatocyte Thawing and Plating Supplements</td>
			<td>Life Technologies</td>
			<td>CM3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary Hepatocyte Maintenance Supplements</td>
			<td>Life Technologies</td>
			<td>CM4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (PBS) pH 7.4</td>
			<td>ThermoFisher scientific</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type 1</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution</td>
			<td>VWR</td>
			<td>45000-717</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of an inflammatory response in primary hepatocyte cultures from mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Histology
Representative light microscopy images of primary isolated and cultured cells are depicted in Figure 1A. Immunocytochemical analysis demonstrates that isolated hepatocytes highly express albumin (red) as well as fibroblast growth factor receptor 4 (FGFR4) (green). Nuclei are stained with 4&#39;,6-diamidino-2-phenylindole (DAPI) (blue). (Figure 1B).
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Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

induction-an-inflammatory-response-primary-hepatocyte-cultures-from

Research

JoVE Journal

Immunology and Infection

9.9K Views.  University of Miami Leonard M. Miller School of Medicine. Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

Video: Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...