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).

Tout l&#39;expérimentation animale doit être effectuée conformément aux réglementations locales et fédérales et avec l&#39;approbation d&#39;une administration institutionnelle IACUC et de sûreté radiologique. 1. Préparation <ol><li> Plusieurs jours avant l&#39;essai, décongeler la bouteille de foie Digest moyenne (LDM) de 500 ml et de recongeler ~ 35 ml aliquotes dans 50 ml tubes coniques. Conserver à -20 ° C jusqu&#39;à ce que nécessaire. </li><li> Un jour avant le test, pré-stérilisation des outils de dissection propres en autoclave. </li><li> Le jour du dosage, la quantité nécessaire de traiter des plaques de culture à 24 puits avec du collagène. <ol><li> Mélanger une partie de 3 mg / ml de collagène de queue de rat avec 50 parties de PBS. Ajouter environ 500 ul à chaque puits d&#39;une plaque de 24 puits et incuber pendant 3-5 min à température ambiante (RT). Retirez le collagène et laisser la plaque sécher à l&#39;air dans le capot. Répétez au moins une fois supplémentaire. Remarque: Un revêtement de collagène peut être effectuée jusqu&#39;à une semaine à l&#39;annonceVance et plaques conservées à 4 ° C scellée dans une pellicule plastique. </li></ol></li><li> Préparer LDM de dosage: Dégel LDM et chaude à 37 ° C et ajuster le pH à 7,4 en utilisant 1 N KOH. Filtre utilisant le filtre de seringue de 0,2 um et placer dans un 42 ° C bain d&#39;eau de recirculation. </li><li> Chaud 25 ml de Foie perfusion du milieu à 42 ° C en recirculation bain d&#39;eau. </li><li> Préparer 90% de silice colloïdale revêtue avec une solution de polyvinylpyrrolidone: Ajouter 1 ml de DPBS 10x à 9 ml de la silice colloïdale revêtue de polyvinylpyrrolidone. Ajuster le pH à 7,4 en utilisant du HCl 0,1N. Filtrer avec un filtre à seringue de 0,2 um stérile. Stocker à température ambiante. </li><li> Chaud Placage moyenne à 37 ° C. </li></ol> 2. Isolement des hépatocytes primaires de souris <ol><li> Mettre en place une pompe péristaltique, les tubes et table de dissection: Stériliser tube par rinçage avec de l&#39;éthanol 5 ml de 70% dans l&#39;eau distillée, puis 10 ml d&#39;eau stérile. Placez les tubes dans le foie perfusion du milieu et de faire fonctionner la pompe à comblertoute la longueur du tube. </li><li> Sacrifiez la souris par la méthode institutionnellement approuvé. </li><li> Disséquer ouvert la cavité abdominale: Vaporiser l&#39;abdomen de souris généreusement avec 70% d&#39;éthanol. Avec des ciseaux extrémités franches, faire une incision médiane à travers le derme de la longueur de l&#39;abdomen et de réfléchir latéralement. Faire une incision similaire dans le péritoine pour exposer les viscères. <ol><li> L&#39;utilisation d&#39;un instrument contondant, déplacer doucement les intestins pour exposer le système vasculaire abdominale Localiser la veine cave inférieure abdominale (IVC) et placer une suture sous le vaisseau sanguin, distale de la veine rénale </li></ol></li><li> Distale par rapport à la suture, placer une aiguille et le cathéter dans la veine cave inférieure, avançant au-delà du niveau de la suture. Avec l&#39;aiguille toujours en place, attachez le fil de suture autour du cathéter pour le maintenir en place. Retirez délicatement l&#39;aiguille. Si fait correctement, le sang avec un débit à travers le cathéter. </li><li> En utilisant une pipette, remplir l&#39;espace restant dans le cathéter avec milieu de perfusion, assurantque l&#39;air est présent. Avec grand soin, fixer le tuyau au cathéter. </li><li> Disséquer la cavité ouverte du poumon: refléter doucement les lobes du foie supérieurs à exposer le diaphragme. Percer délicatement la membrane avec des ciseaux à bout pointu, puis faire une incision latérale pour exposer la cavité pleurale, en prenant soin d&#39;éviter la vésicule biliaire et le système vasculaire pleural. Placez une pince bulldog autour de la veine cave inférieure thoracique juste en amont de la veine hépatique. </li><li> Couper la veine porte et tourner sur la pompe à 3 - 4 ml / min. Perfuser le foie foie avec perfusion du milieu pendant 5 min en utilisant environ 20 ml de milieu de perfusion. Remarque: Le foie doit immédiatement changer du rouge au gris / beige. Si des parties du foie restent en rouge, cela indique probablement une mauvaise perfusion, et la probabilité d&#39;isolement réussi est fortement diminuée. Au cours de la perfusion, soyez prudent pour assurer le support ne fonctionne pas et qu&#39;il n&#39;y a pas de bulles entrent dans le tube. </li><li> Suite à l&#39;incubation 5 min, arrêtla pompe et le transfert de la tubulure à foie Digest moyenne. Redémarrez la pompe et perfuser le foie pendant 10 - 15 min (jusqu&#39;à ce que le milieu est épuisé). </li><li> A la fin de la perfusion, arrêter la pompe. Le foie doit avoir une teinte rosée et semblent quelque peu élargie. Exciser le foie par dissection minutieuse. Retirer la vésicule biliaire et de transférer le foie à 10 cm boîte de culture de tissu. </li><li> Dans une enceinte de sécurité biologique, ajouter 10 ml de placage moyenne (tableau 1) pour le foie. Gratter délicatement le foie en utilisant soit des forceps ou un scalpel pour enlever les hépatocytes. On filtre la suspension en utilisant un tamis cellulaire 100 um et transférer dans un tube conique de 50 ml. Laver la plaque avec 10 ml supplémentaires de milieu d&#39;étalement et la piscine dans le tube conique de 50 ml. </li><li> Pellet les cellules par centrifugation pendant 5 minutes à 350 xg, 4 ° C. </li><li> Milieu de Aspirer et remettre le culot dans 10 ml de milieu d&#39;étalement et ajouter 10 ml de 90% de silice colloïdale revêtue d&#39;polyvinylpyrrolidone. Mélanger délicatement et centrifuger comme dans l&#39;étape 2.11. Après centrifugation, une couche de cellules mortes sera flottant sur le dessus du mélange, tandis que les cellules vivantes se sédimenter au fond. </li><li> Aspirer Les cellules mortes et moyen. Laver 2 fois avec 20 ml de milieu d&#39;étalement, centrifugeuse que dans 2.11. </li><li> Resuspendre les cellules dans 10 ml de milieu d&#39;étalement. Compter les cellules avec un hémocytomètre et placer 9 x 10 4 cellules / puits dans 24 puits des boîtes de culture de collagène traitée. Incuber à 37 ° C une culture tissulaire incubateur pendant 2 heures. Chaque plaque peut être utilisé soit pour le dosage des acides gras oxydation ou l&#39;essai de la lipogénèse. </li><li> Eventuellement, changer puits de milieu d&#39;entretien (tableau 1) et de la culture à 37 ° C. Les cellules peuvent être cultivées pour 2 - 3 jours sans affecter les résultats d&#39;analyse. </li></ol> 3. oxydation des acides gras Assay Avertissement: l&#39;utilisation de la radioactivité peut être dangereux. Tout achat, l&#39;entreposage, la manipulation, et disposal de matières radioactives doit être réalisée en conformité avec, l&#39;état et les règlements et les lignes directrices fédérales institutionnels. <ol><li> 16 - 20 h avant l&#39;essai, laver les cellules 2 fois avec du PBS chaud. Changer les cellules à Sérum famine du milieu sans sérum (tableau 1) avec 20 nM de glucagon et incuber une nuit à 37 ° C. Remarque: En raison sérum fœtal bovin contient une concentration inconnue d&#39;hormones métaboliques (par exemple de l&#39;insuline, le glucagon), le sérum affamer les cellules pour enlever les effets de confusion que cela peut avoir sur le dosage. Les cellules sont viables dans le sérum famine moyen pour&gt; 24 heures. Traitement de glucagon est utilisé pour stimuler l&#39;oxydation des acides gras dans les hépatocytes. </li><li> Le matin de l&#39;essai, préparer la pré-incubation moyenne: Reprendre une quantité appropriée de sodium palmitate dans l&#39;eau ultra pure pour faire une solution 100 mM et de la chaleur à 70 ° C pendant 10 min. Dans le même temps, préparer la quantité requise (0,5 ml par puits d&#39;une plaque de 24 puits)de DMEM avec 25 mM de HEPES, 1% de BSA Fraction V, et 20 nM de glucagon. Chauffer à 37 ° C. <ol><li> Une fois solubilisé, palmitate d&#39;ajouter à une concentration finale de 250 uM dans le milieu. Changer hépatocytes de pré-incubation moyenne et incuber à 37 ° C pendant 2 heures. Réserver une partie de pré-incubation moyenne à 37 ° C pour une utilisation ultérieure. </li></ol></li><li> Au cours de l&#39;incubation, on sèche une quantité appropriée de 14 C-palmitate (0,5 uCi / puits) par evaporation sous azote gazeux. <ol><li> Par exemple, pour mesurer 24 échantillons dans une plaque de 24 puits, transférer 120 ul de 0,1 pCi / ml 14 C-palmitate à un tube de 1,5 ml. Évaporer lentement le solvant par soufflage d&#39;azote gazeux sur la solution à une distance de 3-5 cm dans une hotte éthanol. Le solvant doit s&#39;évaporer à environ 30 - 40 min, en laissant sécher 14 C-palmitate dans le fond du tube. </li></ol></li><li> A environ 15 min avant la fin de l&#39;incubation, le 14 remettre en suspensionC-palmitate dans NaOH 0,1 N (12,5 ul / uCi). Incuber à 70 ° C pendant 10 min. Ajouter trois volumes de chaleur pré-incubation moyenne et mélanger par pipetage de haut en bas. </li><li> De Spike chaque puits avec 25 pi de dilution 14 C-palmitate. Mélanger en secouant délicatement la plaque et incuber à 37 ° C pendant 90 min. Ceci est la plaque d&#39;essai. </li><li> Au cours de l&#39;incubation, préparer la plaque de filtre en papier (Figure 1). <ol><li> Retirez le couvercle d&#39;une plaque de 24 puits stérile. Placer une 2 cm x 2 cm morceau de papier-filtre dans le fond de chacun des puits. Superposez la plaque avec un morceau de 4 &quot;x 7&quot; parafilm. </li><li> En utilisant un grand objet rectangulaire, comme une boîte de pointe de micropipette, frotter le parafilm sur les puits de perforer le parafilm au niveau des ouvertures de puits et de créer un joint sur le reste de la plaque. Retirez les cercles perforés de parafilm couvrant désormais les puits. La plaque devrait maintenant être bien couvert avec du parafilm dans tous les domaines, sauf le bien openings. </li></ol></li><li> 10 min avant la fin de l&#39;incubation, ajouter 200 pi de NaOH 3 N à chaque puits de la plaque de papier filtre, assurant que le papier filtre absorbe la totalité du liquide. <ol><li> Eventuellement avant la congélation, transférer le support à une plaque de 24 puits fraîche. Après avoir lavé une fois avec PBS, la lyse des cellules restantes dans HCl 0,1 N et de calculer la teneur en protéines par dosage BCA. </li></ol></li><li> À la fin de l&#39;incubation, composant logiciel enfichable geler la plaque de dosage dans l&#39;azote liquide. Soyez prudent pour assurer chaque puits est complètement gelé avant de poursuivre. </li><li> Ajouter 100 ul d&#39;acide perchlorique à 70% à chaque puits de la plaque de dosage. Couvrir immédiatement avec la plaque de filtre en papier. Placez les plaques sur un agitateur orbital et de la roche à la vitesse orbitale de 80 min à température ambiante pendant 2 h. </li><li> Après l&#39;incubation, traiter les échantillons: <ol><li> Pour mesurer la fraction de CO 2, le transfert des carrés de papier filtre à 4 ml de fluide de scintillation liquide dans un compteur à scintillationflacon et mesure 14 C signal. </li><li> Pour mesurer la matière soluble dans l&#39;acide, transférer 400 pi de milieu dans un tube de 1,5 ml. Centrifuger à vitesse maximale pendant 10 min. Ajouter 100 ul de surnageant résultant à 500 ul de 2: 1 de chloroforme-méthanol (v / v), et vortex brièvement. <ol><li> Ajouter 250 ul d&#39;eau au mélange, et de nouveau vortex. Centrifuger les échantillons pendant 10 min à 3000 x g. Transfert 200 ul de la phase supérieure à 4 ml de fluide de scintillation liquide dans un flacon à scintillation et la mesure 14 signal C. </li></ol></li></ol></li></ol> 4. lipogenèse Assay <ol><li> Le soir avant de commencer l&#39;essai, se laver les cellules 2 fois avec du PBS chaud. Modifiez les hépatocytes au sérum famine moyenne avec 100 nM d&#39;insuline. Incuber une nuit à 37 ° C. Remarque: En raison sérum fœtal bovin contient une concentration inconnue d&#39;hormones métaboliques (par exemple l&#39;insuline, le glucagon), Le sérum affamer les cellules pour enlever les effets de confusion que cela peut avoir sur le dosage. Les cellules sont viables dans le sérum famine moyen pour&gt; 24 heures. L&#39;insuline est utilisé dans cet essai à stimuler la lipogenèse. </li><li> Faire la lipogénèse moyenne: Sérum famine moyenne avec 100 nM d&#39;insuline, 10 pm acétate froid et 0,5 uCi 3 H-acétate par puits. Changer cellules à la lipogénèse moyen et incuber à 37 ° C pendant 2 heures. Comprennent des composés d&#39;intérêt à être testés. </li><li> Après la période d&#39;incubation, les cellules laver 2 fois avec du PBS. Lyse cellules par grattage dans 120 ul de HCl 0,1. Reserve 10 pi pour le dosage de la protéine (évaluer par dosage BCA), et le transfert de 100 pi à tube de 1,5 ml. </li><li> Extraction des lipides par l&#39;addition de 500 ul de 2: 1 de chloroforme et de methanol (v / v). Vortex brièvement et incuber à température ambiante pendant 5 min. Ajouter 250 ul d&#39;eau, vortex et incuber à température ambiante pendant 5 min de plus. Centrifuger les échantillons 10 min à 3000 xg, RT. Soigneusementtransférer phase inférieure à 4 ml de fluide de scintillation liquide dans un flacon à scintillation et mesurer l&#39;activité de H 3. </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.

Le temps de sacrifice de perfusion doit être inférieur à 3 min pour la perfusion de collagénase et idéal digestion du foie. Une fois la perfusion avec perfusion du milieu est initiée, le foie doit immédiatement changer d&#39;apparence à du rouge à jaune pâle. Après environ 10 minutes d&#39;incubation avec LDM, le foie apparaît enflée et rose. Dans le cas où la perfusion est insuffisante, le foie ne peut pas présenter ces changements, et ce sera typiquement conduire à un rendement plus faible des hépatoc...

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.

Isolements d&#39;hépatocytes entraînent généralement en 1 - 3 x 10 7 cellules totales. Après une nuit d&#39;incubation, les cellules apparaissent hexagonal, dont la plupart seront binucléées (figure 2). Les cellules saines devraient être dépourvu de granulations ou des bulles, qui sont indicatifs de la mort cellulaire. En général, le dosage oxydation des acides gras est exécuté dans trois à quatre répétitions par composé d&#39;essai. Compte pour l...

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

Et la lipogenèse de novo d&#39;acides β oxydation gras constituent des voies métaboliques clés dans des hépatocytes, des voies qui sont perturbées dans plusieurs troubles métaboliques, y compris la maladie du foie gras. Ici, nous démontrons l&#39;isolement des hépatocytes primaires de souris et de décrire la quantification de l&#39;oxydation et de la lipogenèse acide β-gras.

Détermination de oxydation des acides gras et de la lipogenèse dans les hépatocytes primaires de souris

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

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20.1K vues.  University of Massachusetts Medical School.  Et la lipogenèse de novo d'acides β oxydation gras constituent des voies métaboliques clés dans des hépatocytes, des voies qui sont perturbées dans plusieurs troubles métaboliques, y compris la maladie du foie gras. Ici, nous démontrons l'isolement des hépatocytes primaires de souris et de décrire la quantification de l'oxydation et de la lipogenèse acide β-gras. 

Vidéo: 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.

Phosphoprotéomique quantitatives en acide gras Stimulé 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 ...

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Biologie

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.

L&#39;imagerie in vivo et des traitements thérapeutiques dans un modèle de souris orthotopique de cancer des ovaires

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 basé sur la détection de l&#39;acétate de Aldononitrile acide dérivé de glucosamine et de muramique pour la détermination des résidus microbienne dans le sol

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%.

L&#39;isolement et la culture primaire de cellules de foie de rat

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;

Application de l&#39;acide rétinoïque pour obtenir des cultures de ostéocytes de primaires ostéoblastes de souris

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.

Protocole pour l&#39;isolement des hépatocytes humains primaires et correspondant Principales populations de cellules hépatiques non parenchymateuses

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.

Méthodes pour la détermination des taux de glucose et oxydation des acides gras dans le coeur isolé de rat de travail

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.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

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.

Isolement et 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 ...

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.

Isolement des hépatocytes primaires de rat avec contrôle de perfusion multiparamétrique

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