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).

Toda la experimentación con animales debe llevarse a cabo de acuerdo con las regulaciones locales y federales y con la aprobación de una administración institucional IACUC y seguridad radiológica. 1. Preparación <ol><li> Varios días antes del ensayo, descongelar la botella 500 ml de hígado Recopilación Medio (LDM) y vuelva a congelar ~ 35 ml alícuotas en tubos de 50 ml cónicos. Almacenar a -20 ° C hasta que se necesite. </li><li> Un día antes del ensayo, pre-esterilizar herramientas de disección limpia por autoclave. </li><li> En el día del ensayo, el tratamiento de la cantidad necesaria de placas de cultivo de 24 pocillos con colágeno. <ol><li> Mezclar 1 parte de 3 mg / ml de colágeno de cola de rata con 50 partes de PBS. Añadir aproximadamente 500 l a cada pocillo de una placa de 24 pocillos y se incuba durante 3-5 min a temperatura ambiente (RT). Retire el colágeno y permitir que la platina se seque al aire en el capó. Repetir al menos una vez adicional. Nota: recubrimiento de colágeno se puede realizar hasta una semana en anuncioVance y placas almacenadas a 4 ° C sellado en una envoltura de plástico. </li></ol></li><li> Preparar LDM para el ensayo: LDM Descongelar y calentar a 37 ° C y ajustar el pH a 7,4 utilizando 1 N de KOH. Filtrar utilizando filtro de jeringa de 0,2 micras y el lugar en una recirculación de 42 ° C baño de agua. </li><li> Caliente 25 ml de hígado de perfusión del medio a 42 ° C en baño de agua de recirculación. </li><li> Preparar 90% de sílice coloidal recubierto con una solución de polivinilpirrolidona: Añadir 1 ml de 10x DPBS a 9 ml de sílice coloidal recubierto con polivinilpirrolidona. Ajustar el pH a 7,4 usando HCl 0,1 N. Filtro con un filtro de jeringa de 0,2 micras estéril. Guarde a temperatura ambiente. </li><li> Cálido Medio Chapado a 37 ° C. </li></ol> 2. El aislamiento de hepatocitos primarios de ratón <ol><li> Configurar bomba peristáltica, tubos, y mesa de disección: Esterilizar tubo por el lavado con etanol 5 ml 70% en agua destilada, seguido de 10 ml de agua estéril. Coloque la tubería en el hígado de perfusión del medio y de la bomba funcionar para llenartoda la longitud del tubo. </li><li> Sacrificio del ratón por el método institucionalmente aprobado. </li><li> Diseccionar abierta la cavidad abdominal: Rocíe el abdomen del ratón liberalmente con etanol al 70%. Con unas tijeras sin filo, hacer una incisión en la línea media a través de la dermis de la longitud del abdomen y reflejar lateralmente. Haga una incisión similar en el peritoneo para exponer las vísceras. <ol><li> El uso de un instrumento romo, desplazar suavemente los intestinos para exponer la vasculatura abdominal Localizar la vena cava inferior abdominal (IVC) y colocar una sutura por debajo de la de los vasos sanguíneos, distal a la vena renal </li></ol></li><li> Distal a la sutura, colocar una aguja y catéter en la vena cava inferior, avanzando más allá del nivel de la sutura. Con la aguja todavía en su lugar, ate la sutura alrededor del catéter para mantenerlo en su lugar. Retire con cuidado la aguja. Si se hace correctamente, la sangre con el flujo a través del catéter. </li><li> Usando una pipeta, llenar el área restante en el catéter con medio de perfusión, asegurandoque el aire no está presente. Con mucho cuidado, coloque el tubo al catéter. </li><li> Diseccionar abierta la cavidad pulmonar: reflejar suavemente los lóbulos hepáticos superiores para exponer el diafragma. Perfore cuidadosamente el diafragma con tijeras afiladas de punta, a continuación, hacer una incisión lateral para exponer la cavidad pleural, teniendo cuidado de evitar la vesícula biliar y la vasculatura pleural. Coloque una pinza bulldog alrededor de la vena cava inferior torácica proximal a la vena hepática. </li><li> Cortar la vena porta y encienda la bomba a los 3 - 4 ml / min. Perfundir el hígado con hígado de perfusión del medio durante 5 minutos utilizando aproximadamente 20 ml de medio de perfusión. Nota: El hígado debe cambiar inmediatamente de rojo a gris / marrón. Si las porciones del hígado siguen siendo rojo, esto probablemente indica mala perfusión, y la probabilidad de éxito en el aislamiento está muy disminuido. Durante la perfusión, tenga cuidado para asegurar el medio no se ejecuta y que no queden burbujas entran en el tubo. </li><li> Después de la incubación de 5 min, se detienela bomba y la transferencia de la tubería al hígado Digesto Medio. Reinicie la bomba y la perfusión del hígado durante otros 10 - 15 min (hasta que el medio se ha agotado). </li><li> Al final de la perfusión, parar la bomba. El hígado debe tener un tono rosado y parecen algo ampliada. Extirpar el hígado por disección cuidadosa. Eliminar la vesícula biliar y el hígado para transferir 10 cm placa de cultivo tisular. </li><li> En un gabinete de bioseguridad, añadir 10 ml de Revestimiento del Medio (Tabla 1) para el hígado. Raspar suavemente el hígado utilizando fórceps o un bisturí para eliminar los hepatocitos. Filtrar la suspensión utilizando un filtro de células de 100 micras y la transferencia a un tubo cónico de 50 ml. Lavar la placa con un adicional de 10 ml de Revestimiento del Medio y piscina en el tubo cónico de 50 ml. </li><li> Sedimentar las células por centrifugación durante 5 min a 350 xg, 4 ° C. </li><li> Medio Aspirar y pellet se resuspende en 10 ml de Revestimiento del medio y agregar 10 ml de 90% de sílice coloidal recubiertas con polyvinylpyrrolidone. Mezclar suavemente y centrifugar como en el paso 2.11. Después de la centrifugación, una capa de células muertas estará flotando en la parte superior de la mezcla, mientras que las células vivas se sedimenten en el fondo. </li><li> Aspirar las células muertas y medio. Lavar 2 veces con 20 ml de Revestimiento del Medio, centrífuga como en 2.11. </li><li> Resuspender las células en 10 ml de Revestimiento del Medio. Contar las células con un hemocitómetro y coloque 9 x 10 4 células / pocillo en 24 y placas de cultivo de colágeno tratados. Incubar en un 37 ° C incubadora de cultivo de tejidos durante 2 h. Cada placa se puede utilizar ya sea para el ensayo de oxidación de ácidos grasos o en el ensayo lipogénesis. </li><li> Opcionalmente, cambie pozos para Mantenimiento Medio (Tabla 1) y la cultura a 37 ° C. Las células pueden ser cultivadas durante 2 - 3 días sin afectar a los resultados del ensayo. </li></ol> 3. Ensayo de oxidación de ácidos grasos Advertencia: El uso de la radiactividad puede ser peligroso. Todas las compras, el almacenamiento, la manipulación, y dishecho de material radiactivo debe llevarse a cabo de acuerdo con, estatales y las regulaciones y directrices federales institucionales. <ol><li> 16 a 20 hr antes del ensayo, lavar las células 2 veces con PBS caliente. Cambiar las células a suero libre de suero inanición Medio (Tabla 1) con 20 nM de glucagón e incubar durante la noche a 37 ° C. Nota: Debido a que el suero fetal bovino contiene una concentración desconocida de hormonas metabólicas (por ejemplo, insulina, glucagón), suero de hambre las células para eliminar cualquier efecto de confusión que esto puede tener en el ensayo. Las células son viables en suero inanición Medio durante&gt; 24 h. Tratamiento con glucagón se utiliza para estimular la oxidación de ácidos grasos dentro de los hepatocitos. </li><li> En la mañana del ensayo, preparar la pre-incubación Medio: Resuspender una cantidad apropiada de palmitato de sodio en agua ultrapura para preparar una solución 100 mM y se calienta a 70 ° C durante 10 min. Mientras tanto, preparar la cantidad requerida (0,5 ml por pocillo de una placa de 24 pocillos)de DMEM con 25 mM HEPES, 1% BSA Fracción V, y 20 nM de glucagón. Calentar a 37 ° C. <ol><li> Una vez solubilizado, palmitato de añadir a una concentración final de 250 mM al medio. Cambie los hepatocitos de pre-incubación medio y se incuba a 37 ° C durante 2 horas. Reserve algún Pre-Incubación Medio a 37 ° C para su uso posterior. </li></ol></li><li> Durante la incubación, se seca una cantidad apropiada de 14 C-palmitato (0,5 Ci / pocillo) por evaporación bajo gas nitrógeno. <ol><li> Por ejemplo, para medir 24 muestras en una placa de 24 pocillos, transferir 120 l de 0,1 Ci / ml 14 C-palmitato a un tubo de 1,5 ml. Poco a poco se evapora el disolvente etanol soplando gas nitrógeno sobre la solución desde una distancia de 3-5 cm en una campana de humos. El disolvente debe evaporarse en aproximadamente 30 - 40 min, dejando seco 14 C-palmitato en la parte inferior del tubo. </li></ol></li><li> Aproximadamente 15 minutos antes del final de la incubación, resuspender el 14C-Palmitato en NaOH 0,1 N (12,5 l / Ci). Incubar a 70 ° C durante 10 min. Añadir tres volúmenes de tibia Pre-Incubación Medio y mezclar pipeteando arriba y abajo. </li><li> Pico cada pocillo con 25 l de diluirse 14 C-palmitato. Mezclar agitando suavemente la placa e incubar a 37 ° C durante 90 minutos. Esta es la placa de ensayo. </li><li> Durante la incubación, preparar la placa de papel de filtro (Figura 1). <ol><li> Retire la tapa de una placa de 24 pocillos estéril. Coloque un 2 cm x 2 cm trozo de papel de filtro en la parte inferior de cada uno de los pozos. Superposición de la placa con una pieza de 4 &quot;x 7&quot; parafina. </li><li> El uso de un objeto rectangular grande, tal como una caja punta de micropipeta, frotar la parafina en los pozos para perforar la parafina en las aberturas y así crear un sello sobre el resto de la placa. Retire los círculos perforados de parafina ahora cubren los pozos. La placa debe ahora estar bien cubierto con parafilm en todas las áreas, excepto el o bienpenings. </li></ol></li><li> 10 min antes del final de la incubación, añadir 200 l de NaOH 3 N a cada pocillo de la placa de papel de filtro, asegurándose de que el papel de filtro absorbe todo el líquido. <ol><li> Opcionalmente antes de la congelación, transferir el medio de una placa de 24 pocillos fresco. Después de lavar una vez con PBS, lisar las células restantes en HCl 0,1 N y calcular el contenido de proteína mediante ensayo BCA. </li></ol></li><li> Al final de la incubación, complemento congelar la placa de ensayo en nitrógeno líquido. Tenga cuidado para asegurar así cada uno está completamente congelado antes de proceder. </li><li> Añadir 100 l de 70% ácido perclórico a cada pocillo de la placa de ensayo. Inmediatamente cubrir con el plato de papel de filtro. Coloque las placas en un agitador orbital y el rock a la velocidad orbital de 80 rpm a temperatura ambiente durante 2 horas. </li><li> Después de la incubación, procesar las muestras: <ol><li> Para medir la fracción de CO 2, la transferencia de los cuadrados de papel de filtro a 4 ml de fluido de centelleo líquido en un centelleovial y la medida 14 de la señal C. </li><li> Para medir el material soluble ácido, transferir 400 l de medio a un tubo de microcentrífuga de 1,5 ml. Centrifugar a máxima velocidad durante 10 min. Añadir 100 l del sobrenadante resultante a 500 l de 2: 1 cloroformo-metanol (v / v), y agitar brevemente. <ol><li> Añadir 250 l de agua a la mezcla, y agitar de nuevo. Centrifugar las muestras durante 10 min a 3000 x g. Transferencia de 200 l de la fase superior a 4 ml de fluido de centelleo líquido en un vial de centelleo y medir 14 la señal C. </li></ol></li></ol></li></ol> 4. lipogénesis Ensayo <ol><li> La noche antes de comenzar el ensayo, se lavan las células 2 veces con PBS caliente. Cambie los hepatocitos a suero inanición Medio con 100 nM de insulina. Incubar durante la noche a 37 ° C. Nota: Debido a que el suero fetal bovino contiene una concentración desconocida de hormonas metabólicas (por ejemplo, insulina, glucagón), Suero de comer a las células para eliminar cualquier efecto de confusión que esto puede tener en el ensayo. Las células son viables en suero inanición Medio durante&gt; 24 h. La insulina se usa en este ensayo para estimular la lipogénesis. </li><li> Hacer lipogénesis Medio: Suero inanición medio con 100 nM de insulina, 10 mM de acetato de frío y 0,5 Ci 3 H-Acetato por pocillo. Cambie células a lipogénesis Medium y se incuba a 37 ° C durante 2 hr. Incluir todos los compuestos de interés para ser probados. </li><li> Después de que el período de incubación, lavar las células 2 veces con PBS. Células Lyse por raspado en 120 l de HCl 0,1 N. Reserva 10 l para el ensayo de proteínas (evaluar por ensayo BCA), y la transferencia de 100 l a tubo de microcentrífuga de 1,5 ml. </li><li> Lípidos extracto por adición de 500 l de 2: 1 cloroformo-metanol (v / v). Vortex brevemente e incubar a temperatura ambiente durante 5 min. Añadir 250 l de agua, vortex e incubar a temperatura ambiente durante 5 min. Centrifugar las muestras 10 min a 3.000 xg, RT. Cuidadosamentetransferencia de fase inferior a 4 ml de fluido de centelleo líquido en un vial de centelleo y medir la actividad 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.

El tiempo desde el sacrificio a la perfusión debe ser inferior a 3 min para la perfusión ideal y digestión con colagenasa del hígado. Una vez que se inicia la perfusión con perfusión del medio, el hígado debe cambiar inmediatamente la apariencia de de rojo a pálido. Después de aproximadamente 10 minutos de incubación con LDM, el hígado aparecerá hinchada y de color rosa. En el caso de que la perfusión es insuficiente, el hígado no puede exhibir estos cambios, y esto normalmente dará como resultado un rend...

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.

Aislamientos de hepatocitos suelen dar en 1 - 3 x 10 7 células totales. Después de la incubación durante la noche, las células van a aparecer hexagonal, muchos de los cuales se binucleadas (Figura 2). Las células sanas deben estar desprovisto de granulaciones o vesículas, que son indicativos de la muerte celular. En general, el ensayo de oxidación de ácidos grasos se ejecuta en tres o cuatro repeticiones por compuesto de ensayo. Condes de las CO 2 mue...

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 la lipogénesis de novo y la oxidación de ácidos grasos β-constituyen vías metabólicas claves en los hepatocitos, las vías que son perturbados en varios trastornos metabólicos, incluyendo la enfermedad de hígado graso. Aquí se demuestra el aislamiento de hepatocitos primarios de ratón y describimos cuantificación de la oxidación de ácidos grasos β-y la lipogénesis.

Determinación de la oxidación de ácidos grasos y la lipogénesis en el ratón hepatocitos primarios

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 vistas.  University of Massachusetts Medical School.  De la lipogénesis de novo y la oxidación de ácidos grasos β-constituyen vías metabólicas claves en los hepatocitos, las vías que son perturbados en varios trastornos metabólicos, incluyendo la enfermedad de hígado graso. Aquí se demuestra el aislamiento de hepatocitos primarios de ratón y describimos cuantificación de la oxidación de ácidos grasos β-y la lipogénesis. 

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.

Phosphoproteomics cuantitativo de ácidos grasos estimulada 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 ...

Investigación

Biología

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.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

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-basado en la detección de ácido Aldononitrile acetato derivado de glucosamina y murámico de la determinación de residuos microbiana en el suelo

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%.

Aislamiento y cultivo primario de células hepáticas de ratas

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;

La aplicación de ácido retinoico para obtener cultivos de osteocitos principal del ratón osteoblastos

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.

Protocolo para el aislamiento de hepatocitos primarios humanos y la correspondiente principales poblaciones de células hepáticas no parenquimatosas

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.

Los métodos para la determinación de las tasas de glucosa y la oxidación de ácidos grasos en el corazón de rata aislado de Trabajo

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.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

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.

Aislamiento y cultivo de sándwich de colágeno 3D de hepatocitos primarios de ratón para estudiar el papel del citoesqueleto en la formación canalicular de bilis en 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.

Aislamiento de hepatocitos primarios de rata con control de perfusión multiparamétrico

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