We would like to acknowledge Susan Gray and Umadevi Chalasani for their help with technical aspects of the hepatocyte isolation protocol. This work was supported by NIDDK grant 5R01DK089185 (to M.P. Cooper) and the DERC Pilot and Feasibility Program at UMMS (to M.P. Cooper).

Tutti sperimentazione animale deve essere effettuata in conformità con le normative locali e federali e con l&#39;approvazione di un IACUC e radioprotezione amministrazione istituzionale. 1. Preparazione <ol><li> Alcuni giorni prima del test, scongelare il flacone da 500 ml di fegato Digest Medium (LDM) e ricongelare ~ 35 ml aliquote in 50 ml provette coniche. Conservare a -20 ° C fino al momento dell&#39;uso. </li><li> Un giorno prima del test, pre-sterilizzare strumenti di dissezione puliti in autoclave. </li><li> Il giorno del test, trattare la quantità necessaria di piastre di coltura a 24 pozzetti con collagene. <ol><li> Mescolare 1 parte di 3 mg / ml di collagene coda di ratto con 50 parti di PBS. Aggiungere circa 500 ml in ciascun pozzetto di una piastra da 24 pozzetti e incubare per 3-5 minuti a temperatura ambiente (RT). Rimuovere il collagene e lasciare asciugare la piastra all&#39;aria in cappuccio. Ripetere almeno una volta in più. Nota: Rivestimento collagene può essere effettuata fino a una settimana a annuncioVance e piatti conservati a 4 ° C sigillato in involucro di plastica. </li></ol></li><li> Preparare LDM per l&#39;analisi: Scongelare LDM e caldo a 37 ° C e aggiustare il pH a 7,4 con 1 N KOH. Filtro utilizzando 0,2 micron siringa filtro e posto in un ricircolo 42 ° C bagnomaria. </li><li> Caldo 25 ml di fegato di perfusione medio a 42 ° C a bagnomaria. Ricircolo </li><li> Preparare 90% di silice colloidale con una soluzione di polivinilpirrolidone rivestita: Aggiungere 1 ml di DPBS 10x a 9 ml di silice colloidale rivestite con polivinilpirrolidone. Regolare il pH a 7,4 con 0,1 N HCl. Filtrare con un filtro a siringa da 0,2 micron sterile. Conservare a temperatura ambiente. </li><li> Caldo placcatura medio a 37 ° C. </li></ol> 2. Isolamento di mouse primario Epatociti <ol><li> Impostare pompa peristaltica, tubi, e la tabella dissezione: Sterilizzare tubi da vampate di calore con 5 ml etanolo al 70% in acqua distillata, seguita da 10 ml di acqua sterile. Mettere il tubo in fegato perfusione Medium e pompa eseguire per riempireintera lunghezza del tubo. </li><li> Sacrifica il mouse con il metodo istituzionalmente approvato. </li><li> Sezionare aperto della cavità addominale: Spruzzare l&#39;addome del mouse liberamente con il 70% di etanolo. Utilizzando le forbici blunt-end, fare una incisione mediana attraverso il derma la lunghezza dell&#39;addome e riflettono lateralmente. Eseguire un&#39;incisione simile nel peritoneo per esporre le viscere. <ol><li> Utilizzando un corpo contundente, spostare dolcemente gli intestini per esporre la vascolarizzazione addominale Individuare il addominale vena cava inferiore (IVC) e posizionare una sutura sotto il vaso sanguigno, distale alla vena renale </li></ol></li><li> Distale alla sutura, posizionare un ago e catetere nel IVC, farlo avanzare oltre il livello della sutura. Con l&#39;ago ancora nel luogo, cravatta la sutura intorno al catetere per tenerlo in posizione. Rimuovere con cautela l&#39;ago. Se fatto correttamente, il sangue con flusso attraverso il catetere. </li><li> Usando una pipetta, riempire l&#39;area rimanente nel catetere con mezzo di perfusione, assicurandoche l&#39;aria non sia presente. Con grande cura, collegare il tubo al catetere. </li><li> Sezionare aperto la cavità polmonare: riflettere delicatamente i lobi del fegato superiori per esporre il diaframma. Forare con attenzione il diaframma con le forbici affilate punte, poi fare una incisione laterale per esporre la cavità pleurica, avendo cura di evitare la cistifellea e vascolare pleurico. Posizionare un morsetto bulldog intorno al torace vena cava inferiore solo prossimale alla vena epatica. </li><li> Tagliare la vena porta e accendere la pompa a 3 - 4 ml / min. Profumato fegato con fegato di perfusione medio per 5 minuti con circa 20 ml di terreno di perfusione. Nota: Il fegato deve cambiare immediatamente dal rosso al grigio / marrone chiaro. Se porzioni di fegato rimangono rossa, questo probabilmente indica scarsa perfusione, e la probabilità di isolamento di successo è notevolmente diminuita. Durante la perfusione, essere attenti a garantire il supporto non si esaurisce e che bolle entrano nel tubo. </li><li> Dopo la 5 min di incubazione, fermatala pompa e il tubo di trasferimento Fegato Digest Medium. Riavviare la pompa e perfusione del fegato per altri 10 - 15 minuti (finché il terreno è esaurito). </li><li> Al termine della perfusione, fermare la pompa. Il fegato dovrebbe avere un colore rosato e apparire in qualche modo allargata. Asportare il fegato da un&#39;attenta dissezione. Rimuovere la cistifellea e trasferire il fegato a 10 centimetri piatto di coltura di tessuti. </li><li> In un armadio biosicurezza, aggiungere 10 ml di placcatura medio (Tabella 1) al fegato. Raschiare delicatamente il fegato utilizzando sia pinze o un bisturi per rimuovere le epatociti. Filtrare la sospensione con un colino cella di 100 micron e trasferimento in una provetta da 50 ml. Lavare la piastra con altri 10 ml di placcatura medio e piscina in tubo conico da 50 ml. </li><li> Agglomerare le cellule per centrifugazione per 5 minuti a 350 xg, a 4 ° C. </li><li> Aspirare a medio e risospendere pellet in 10 ml di placcatura medio e aggiungere 10 ml di 90% di silice colloidale rivestito con Polyvinylpyrrolidone. Mescolare delicatamente e centrifugare come al punto 2.11. Dopo centrifugazione, uno strato di cellule morte sarà galleggiano sulla superficie della miscela, mentre le cellule vive si sedimentare sul fondo. </li><li> Aspirare le cellule morte e le medie. Lavare 2 volte con 20 ml di placcatura Medium, centrifuga come a 2.11. </li><li> Risospendere le cellule in 10 ml di placcatura Media. Contare le celle con un emocitometro e posizionare 9 x 10 4 cellule / pozzetto in 24 pozzetti piatti collagene trattati cultura. Incubare a 37 ° C tessuto cultura incubatore per 2 ore. Ogni piastra può essere utilizzata sia per il saggio Fatty Acid ossidazione o il dosaggio Lipogenesi. </li><li> Facoltativamente, modificare pozzetti Manutenzione Medium (Tabella 1) e cultura a 37 ° C. Le celle possono essere coltivate per 2 - 3 giorni senza influenzare i risultati del test. </li></ol> 3. acidi grassi ossidazione Assay Attenzione: L&#39;uso di radioattività può essere pericoloso. Tutti gli acquisti, lo stoccaggio, la movimentazione, e disposal di materiale radioattivo deve essere effettuata nel rispetto, statali e regolamenti e linee guida federali istituzionali. <ol><li> 16 - 20 ore prima del dosaggio, lavare le cellule 2 volte con PBS caldo. Modificare le cellule di siero Starvation mezzo privo di siero (Tabella 1) con 20 nM glucagone e incubare una notte a 37 ° C. Nota: Poiché siero fetale bovino contiene una concentrazione sconosciuta di ormoni metabolici (per esempio insulina, glucagone), siero morire di fame le cellule per rimuovere eventuali effetti confondenti questa può avere sul dosaggio. Le cellule sono vitali nel siero fame medio per&gt; 24 ore. Trattamento glucagone è usato per stimolare ossidazione degli acidi grassi all&#39;interno epatociti. </li><li> La mattina del saggio, preparare preincubazione medio: Risospendere una quantità appropriata di sodio palmitato in acqua ultrapura per ottenere una soluzione 100 mM e si riscalda a 70 ° C per 10 min. Nel frattempo, preparare la quantità necessaria (0,5 ml per pozzetto di una piastra da 24 pozzetti)di DMEM con 25 mM HEPES, 1% BSA Frazione V, e 20 nM glucagone. Riscaldare a 37 ° C. <ol><li> Una volta solubilizzato, palmitato aggiungere ad una concentrazione finale di 250 mM al mezzo. Cambiare epatociti di pre-incubazione medio e incubare a 37 ° C per 2 ore. Prenota alcuni pre-incubazione medio a 37 ° C per un uso successivo. </li></ol></li><li> Durante l&#39;incubazione, asciugare una quantità appropriata di 14 C-palmitato (0,5 pCi / pozzetto) per evaporazione sotto azoto. <ol><li> Ad esempio, per misurare 24 campioni in una piastra a 24 pozzetti, trasferire 120 ml di 0,1 pCi / ml 14 C-palmitato in una provetta da 1,5 ml. Lentamente si evapora il solvente etanolo soffiando gas azoto sulla soluzione da una distanza di 3-5 cm in una cappa aspirante. Il solvente deve evaporare in circa 30 - 40 min, lasciando asciutta 14 C-palmitato nel fondo della provetta. </li></ol></li><li> Circa 15 minuti prima della fine dell&#39;incubazione, risospendere il 14C-palmitato in NaOH 0,1 N (12.5 ml / uCi). Incubare a 70 ° C per 10 min. Aggiungere tre volumi di caldo pre-incubazione medio e mescolare pipettando su e giù. </li><li> Spike ogni pozzetto con 25 ml di diluito 14 C-palmitato. Mescolare scuotendo delicatamente la piastra e incubare a 37 ° C per 90 min. Questa è la piastra saggio. </li><li> Durante l&#39;incubazione, preparare la piastra di filtraggio di carta (Figura 1). <ol><li> Togliere il coperchio da un 24-pozzetti sterili. Posizionare un 2 cm x 2 cm pezzo di carta da filtro sul fondo di ciascun pozzetto. Sovrapporre il piatto con un pezzo di 4 &quot;x 7&quot; parafilm. </li><li> Utilizzando un grande oggetto rettangolare, ad esempio una casella punta della micropipetta, strofinare la parafilm sui pozzetti per perforare la parafilm le aperture oltre e creare una tenuta sul resto della piastra. Rimuovere i cerchi forati di parafilm ora coprono i pozzetti. La piastra dovrebbe essere ben coperto con parafilm in tutte le zone tranne il bene openings. </li></ol></li><li> 10 min prima della fine dell&#39;incubazione, aggiungere 200 ml di NaOH N 3 a ciascun pozzetto della piastra Filter Paper, assicurandosi che la carta da filtro assorbe tutto il liquido. <ol><li> Facoltativamente prima del congelamento, trasferire il mezzo di una piastra da 24 pozzetti fresco. Dopo aver lavato una volta con PBS, la lisi dei rimanenti cellule in 0,1 N HCl e calcolare il contenuto proteico mediante test BCA. </li></ol></li><li> Al termine dell&#39;incubazione, snap-congelare la piastra di dosaggio in azoto liquido. Fare attenzione a garantire che ogni bene è completamente congelato prima di procedere. </li><li> Aggiungere 100 ml di 70% di acido perclorico in ogni pozzetto della piastra saggio. Coprire immediatamente con la piastra di carta da filtro. Posizionare le piastre su un agitatore orbitale e roccia alla velocità orbitale di 80 rpm a temperatura ambiente per 2 ore. </li><li> In seguito l&#39;incubazione, processare i campioni: <ol><li> Per misurare la frazione di CO 2, trasferire i quadrati di carta filtro a 4 ml di fluido di scintillazione liquida in un scintillazionefiala e misura 14 Segnale C. </li><li> Per misurare il materiale solubile acido, trasferire 400 ml di mezzo in una provetta da 1,5 ml microcentrifuga. Centrifugare alla massima velocità per 10 min. Aggiungere 100 pl del supernatante risultante a 500 ml di 2: 1 cloroformio-metanolo (v / v) e miscelare brevemente. <ol><li> Aggiungere 250 ml di acqua alla miscela, e di nuovo vortice. Campioni di centrifugazione per 10 min a 3000 x g. Trasferimento 200 ml di fase superiore a 4 ml di liquido di scintillazione liquido in un flaconcino scintillazione e misura 14 Segnale C. </li></ol></li></ol></li></ol> 4. Lipogenesi Assay <ol><li> La sera prima di iniziare il test, lavare le cellule 2 volte con il caldo PBS. Modificare le epatociti a Serum fame media con 100 nM insulina. Incubare una notte a 37 ° C. Nota: Poiché siero fetale bovino contiene una concentrazione sconosciuta di ormoni metabolici (per esempio insulina, glucagone), Il siero morire di fame le cellule per rimuovere eventuali effetti confondenti questa può avere sul dosaggio. Le cellule sono vitali nel siero fame medio per&gt; 24 ore. L&#39;insulina è usato in questo test per stimolare la lipogenesi. </li><li> Fai Lipogenesi Media: Siero fame medio con 100 nM insulina, 10 micron acetato freddo e 0,5 uCi 3 H-acetato per pozzetto. Modificare le celle a Lipogenesi media e incubare a 37 ° C per 2 ore. Includere eventuali composti di interesse da testare. </li><li> Dopo il periodo di incubazione, lavare le cellule 2 volte con PBS. Lisare le cellule raschiando in 120 ml di 0,1 N HCl. Riserva 10 microlitri per l&#39;analisi delle proteine ​​(valutare mediante test BCA), e il trasferimento di 100 l per provetta da 1,5 ml microcentrifuga. </li><li> Estratto lipidi per aggiunta di 500 ml di 2: 1 cloroformio-metanolo (v / v). Vortex brevemente e incubare a temperatura ambiente per 5 min. Aggiungere 250 ml di acqua, vortice e incubare a temperatura ambiente per un ulteriore 5 min. Centrifugare i campioni 10 min a 3000 x g, RT. Accuratamentetrasferire fase inferiore a 4 ml liquido di scintillazione liquido in un flaconcino scintillazione e misurare l&#39;attività 3 H. </li></ol>

<ol>
	<li>Clark, J. M., Brancati, F. L., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+and+etiology+of+elevated+aminotransferase+levels+in+the+United+States.">The prevalence and etiology of elevated aminotransferase levels in the United States.</a> The American journal of gastroenterology. 98, 960-967 (2003).</li><li>Lazo, 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=Prevalence+of+nonalcoholic+Fatty+liver+disease+in+the+United+States:+the+third+national+health+and+nutrition+examination+survey,+1988-1994.">Prevalence of nonalcoholic Fatty liver disease in the United States: the third national health and nutrition examination survey, 1988-1994.</a> American journal of epidemiology. 178, 38-45 (2013).</li><li>Angulo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonalcoholic+fatty+liver+disease.">Nonalcoholic fatty liver disease.</a> The New England journal of medicine. 346, 1221-1231 (2002).</li><li>Adams, L. 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=The+natural+history+of+nonalcoholic+fatty+liver+disease:+a+population-based+cohort+study.">The natural history of nonalcoholic fatty liver disease: a population-based cohort study.</a> Gastroenterology. 129, 113-121 (2005).</li><li>Feldstein, A. 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=Hepatocyte+apoptosis+and+fas+expression+are+prominent+features+of+human+nonalcoholic+steatohepatitis.">Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis.</a> Gastroenterology. 125, 437-443 (2003).</li><li>Day, C. P., James, O. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steatohepatitis:+a+tale+of+two+'hits'.">Steatohepatitis: a tale of two 'hits'.</a> Gastroenterology. 114, 842-845 (1998).</li><li>Donnelly, K. L., 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=Sources+of+fatty+acids+stored+in+liver+and+secreted+via+lipoproteins+in+patients+with+nonalcoholic+fatty+liver+disease.">Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease.</a> The Journal of clinical investigation. 115, 1343-1351 (2005).</li><li>Lambert, J. E., Ramos-Roman, M. A., Browning, J. D., Parks, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+de+novo+lipogenesis+is+a+distinct+characteristic+of+individuals+with+nonalcoholic+fatty+liver+disease.">Increased de novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease.</a> Gastroenterology. 146, 726-735 (2014).</li><li>Parks, E. J., Hellerstein, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+patient-oriented+research.+Recent+advances+in+liver+triacylglycerol+and+fatty+acid+metabolism+using+stable+isotope+labeling+techniques.">Thematic review series: patient-oriented research. Recent advances in liver triacylglycerol and fatty acid metabolism using stable isotope labeling techniques.</a> Journal of lipid research. 47, 1651-1660 (2006).</li><li>Befroy, D. 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=Direct+assessment+of+hepatic+mitochondrial+oxidative+and+anaplerotic+fluxes+in+humans+using+dynamic+13C+magnetic+resonance+spectroscopy.">Direct assessment of hepatic mitochondrial oxidative and anaplerotic fluxes in humans using dynamic 13C magnetic resonance spectroscopy.</a> Nature medicine. 20, 98-102 (2014).</li><li>Goncalves, L. A., Vigario, A. M., Penha-Goncalves, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+isolation+of+murine+hepatocytes+for+in+vitro+malaria+liver+stage+studies.">Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.</a> Malaria journal. 6, 169 (2007).</li></ol>

The authors indicate they have no conflicts of interest.

Il tempo dal sacrificio perfusione deve essere inferiore a 3 minuti per perfusione ideale e collagenasi digestione del fegato. Una volta iniziata la perfusione con perfusione Media, il fegato dovrebbe cambiare immediatamente aspetto a dal rosso al pallido. Dopo circa 10 minuti di incubazione con LDM, il fegato apparirà gonfio e rosa. Nel caso in cui perfusione è insufficiente, il fegato non può presentare tali modifiche, e ciò avrà come risultato una resa minore epatociti. Dopo le fasi ...

Non-alcoholic fatty liver disease is one of the leading causes of liver disease in Westernized cultures1,2. Lipid accumulation within the liver is associated with cell death, fibrosis, and liver failure via yet unknown mechanisms3-6. In fatty liver disease, hepatocyte-mediated &#946;-fatty acid oxidation and de novo lipogenesis are important determinants of net lipid accumulation7,8. This article will, therefore, focus on hepatocyte isolation, followed by quantification of &#946;-fatty acid oxidation and de novo lipogenesis.
Numerous methodologies have been developed to interrogate hepatocyte lipid metabolism. Though it is possible to measure metabolism of fat in vivo using stable isotopes9,10, these methods are costly, and require large numbers of animals. Additionally, the ability to investigate the effect of exogenous chemicals is limited due to the nature of in vivo experimentation. In contrast, the isolation of primary hepatocytes from mouse liver provides an affordable avenue to pursue11. Furthermore, studying hepatocytes in culture allows investigators to study the effects of varying chemicals on lipid processing while circumventing the difficulties of in vivo experimentation. Finally, isolated hepatocytes avoid any confounding from varying genetics since they are derived from the liver of a single animal.
Here we isolate and culture of hepatocytes, and we measure &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled palmitate. The protocol detailed below is straight forward, effective, and reproducible.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Liver Perfusion Medium</td>
			<td>Life Technologies</td>
			<td>17701038</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Digest Medium</td>
			<td>Life Technologies</td>
			<td>17703034</td>
			<td>Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>10X DPBS</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>26140079&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>Life Technologies</td>
			<td>A1048301&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Colloidal silica coated with polyvinylpyrrolidone</td>
			<td>GE Life Sciences</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Cellgro</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / Streptomycin</td>
			<td>Cellgro</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I0516-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin (BSA), Fraction V</td>
			<td>MP Biomedicals</td>
			<td>103703</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Culture Dish</td>
			<td>Corning Falcon</td>
			<td>353047&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon S3 Tubing&#160;</td>
			<td>Cole Parmer</td>
			<td>06460-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Leur Lock to 200 Barb Connectors</td>
			<td>Cole Parmer</td>
			<td>45518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>24G x 3/4&#34; Catheter</td>
			<td>SurFlo</td>
			<td>SROX2419CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Perma-Hand Silk Suture</td>
			<td>Ethicon</td>
			<td>683G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Corning Falcon</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>IsoTemp 3013HD Recirculating Water Bath</td>
			<td>Fisher</td>
			<td>13-874-3</td>
			<td></td>
		</tr>
		<tr>
			<td>MasterFlex C/L Peristaltic Pump</td>
			<td>MasterFlex</td>
			<td>HV-77122-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Microclamp</td>
			<td>Roboz</td>
			<td>RS-7438</td>
			<td>Pre-sterilize in autoclave</td>
		</tr>
		<tr>
			<td>5&#8221; Straight, Blunt-Blunt Operating Scissors</td>
			<td>Roboz</td>
			<td>RS-6810</td>
			<td>Pre-sterilize in autoclave</td>
		</tr>
		<tr>
			<td>24mm Blade Straight, Sharp-point Microdissecting Scissors</td>
			<td>Roboz</td>
			<td>RS-5912</td>
			<td>Pre-sterilize in autoclave</td>
		</tr>
		<tr>
			<td>4&#8221; 0.8mm Tip Microdissecting Forceps</td>
			<td>Roboz</td>
			<td>RS-5130</td>
			<td>Pre-sterilize in autoclave</td>
		</tr>
		<tr>
			<td>4&#8221; 0.8mm Tip Full Curve Microdissecting Forceps</td>
			<td>Roboz</td>
			<td>RS-5137</td>
			<td>Pre-sterilize in autoclave</td>
		</tr>
		<tr>
			<td>60 mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Corning Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000, 200, 20 &#956;L pipet and tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Isolamenti epatociti tipicamente risultato in 1 - 3 x 10 7 cellule totali. Dopo incubazione per una notte, le cellule appariranno esagonale, molti dei quali saranno binucleate (Figura 2). Le cellule sane dovrebbero essere privi di granulazioni o vesciche, indicativi della morte cellulare. In generale, dosaggio di acidi grassi ossidazione viene eseguito in tre o quattro repliche per composto in esame. Conti per le CO 2 campioni sono circa un quinto di qu...

Watch this Scientific Journal Video about Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes at JoVE.com

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

De novo lipogenesi e acido ossidazione β-grassi costituiscono importanti vie metaboliche in epatociti, percorsi che sono perturbate in diversi disordini metabolici, tra cui malattia del fegato grasso. Qui mostriamo isolamento di epatociti primari di topo e descriviamo la quantificazione di ossidazione degli acidi β-grassi e lipogenesi.

Determinazione della ossidazione degli acidi grassi e la lipogenesi nel topo primaria Epatociti

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

determination-fatty-acid-oxidation-lipogenesis-mouse-primary

Research

JoVE Journal

Biology

20.1K Views.  University of Massachusetts Medical School.  De novo lipogenesi e acido ossidazione β-grassi costituiscono importanti vie metaboliche in epatociti, percorsi che sono perturbate in diversi disordini metabolici, tra cui malattia del fegato grasso. Qui mostriamo isolamento di epatociti primari di topo e descriviamo la quantificazione di ossidazione degli acidi β-grassi e lipogenesi. 

Video: Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Fosfoproteomica quantitativa in acidi grassi Stimolati Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Ricerca

Biologia

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

GC-based Detection di acetato derivatizzata Glucosamina e muramico Acid Aldononitrile per la determinazione dei residui microbica nel terreno

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolamento e la coltura primaria di cellule epatiche di ratto

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

Applicazione di acido retinoico per ottenere colture osteocytes da principale del mouse osteoblasti

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Protocollo per l&#39;isolamento di epatociti umani primari e corrispondenti principali popolazioni di cellule del fegato non-parenchimali

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

Metodi per la determinazione dei tassi di glucosio e acidi grassi ossidazione nel cuore isolato di lavoro Rat

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e cultura dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

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.

Isolamento e 3D Collagen Sandwich Cultura del topo primario Epatociti per studiare il ruolo del citoscheletro nella formazione canalicolare bile 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 ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Isolamento degli epatociti primari di ratto con controllo della perfusione multiparametrica

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...