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).

Alle Tierversuche sollten in Übereinstimmung mit den örtlichen und nationalen Vorschriften und mit der Zustimmung eines institutionellen IACUC und Strahlungssicherheit Verwaltung durchgeführt werden. 1. Vorbereitung <ol><li> Einige Tage vor dem Test, tauen die 500 ml Flasche Liver Digest Medium (LDM) und wieder einfrieren ~ 35 ml Aliquots in 50 ml konischen Röhrchen. Lagerung bei -20 ° C, bis sie benötigt. </li><li> Einen Tag vor dem Assay, pre-sterilisiert sauberen Dissektion Tools von Autoklaven. </li><li> Am Tag des Assays, behandeln die notwendige Menge von 24-Well-Kulturplatten mit Collagen. <ol><li> 1 Teil von 3 mg / ml Rattenschwanzkollagen mit 50 Teilen PBS. In etwa 500 &amp; mgr; l in jede Vertiefung einer 24-Well-Platte und Inkubation für 3-5 Minuten bei Raumtemperatur (RT). Entfernen Sie die Kollagen und lassen Sie die Platte trocken in der Haube in die Luft. Wiederholen wenigstens ein weiteres Mal. Hinweis: Collagen-Beschichtung kann bis zu einer Woche in ad durchgeführt werdenVance und Platten bei 4 ° C in Plastikfolie abgedichtet gelagert. </li></ol></li><li> Bereiten LDM für Assay: Thaw LDM und warm bis 37 ° C und pH-Wert auf 7,4 mit 1 N KOH. Filter mit 0,2 um Spritzenfilter und in einen Umluft 42 ° C Wasserbad. </li><li> Warm 25 ml Leberperfusion Medium auf 42 ° C im Umluft Wasserbad. </li><li> Vorbereitung 90% kolloidales Siliciumdioxid, das mit Polyvinylpyrrolidon-Lösung überzogen: 1 ml 10x DPBS zu 9 ml kolloidaler Kieselsäure mit Polyvinylpyrrolidon beschichtet. Einstellen des pH auf 7,4 unter Verwendung von 0,1 N HCl. Filter mit einer sterilen 0,2 &amp; mgr; m Spritzenfilter filtriert. Lagern bei RT. </li><li> Warm Plating Medium auf 37 ° C. </li></ol> 2. Isolierung von Primär Maushepatozyten <ol><li> Einrichten peristaltische Pumpe, Schläuche und Dissektionstisch: Sterilisieren Schlauch durch Spülen mit 5 ml 70% Ethanol in destilliertem Wasser, gefolgt von 10 ml sterilem Wasser. Zeigen Schlauch in Leberperfusion Medium und führen Pumpe zu füllengesamte Rohrlänge. </li><li> Opfern Maus durch die institutionell genehmigten Methode. </li><li> Sezieren offen die Bauchhöhle: Sprühen Sie die Mausunterleib zügig mit 70% Ethanol. Verwendung blunt-end Schere, einen Mittelschnitt durch die Dermis die Länge der Bauch und seitlich reflektieren. Einen ähnlichen Schnitt in der Bauchfell, um die Eingeweide aus. <ol><li> Mit einem stumpfen Gegenstand vorsichtig zu verdrängen den Darm zu entlarven die Bauchgefäß Suchen Sie die Bauch untere Hohlvene (IVC) und legen Sie eine Naht unter dem Blutgefäß, distal der Nierenvenen </li></ol></li><li> Distal des Nahtmaterials Platzieren einer Nadel und der Katheter in die IVC, Vorschieben über das Niveau des Fadens. Mit der Nadel noch vorhanden, binden die Naht um den Katheter, um es in Position zu halten. Die Nadel vorsichtig entfernen. Wenn es richtig mit Fluss durch den Katheter durchgeführt, Blut. </li><li> Unter Verwendung einer Pipette, füllen Sie die restliche Fläche in dem Katheter mit Perfusionsmedium, sicherzustellen,daß keine Luft vorhanden ist. Mit großer Sorgfalt, befestigen Sie den Schlauch mit dem Katheter. </li><li> Sezieren offen die Lunge Hohlraum: sanft spiegeln die überlegene Leberlappen, um die Membran freizulegen. Sorgfältig durchstechen die Membran mit scharfen Spitze Schere, dann machen Sie eine laterale Inzision, um die Brusthöhle freizulegen, kümmert sich um die Gallenblase und Pleura Gefäßsystem zu vermeiden. Legen Sie eine Bulldogge Schelle um den Brust untere Hohlvene gerade proximal der Lebervene. </li><li> Schneiden Sie die Pfortader und schalten Sie die Pumpe mit einem 3 - 4 ml / min. Perfusion der Leber Leberperfusion Medium für 5 min unter Verwendung von etwa 20 ml Perfusionsmedium. Hinweis: Die Leber sollten sofort von Rot zu ändern bis grau / tan. Wenn Teile der Leber rot bleiben, dürfte dies deutet auf eine schlechte Durchblutung, und die Wahrscheinlichkeit einer erfolgreichen Isolierung stark vermindert. Während der Perfusion, seien Sie vorsichtig, um das Medium nicht auslaufen zu gewährleisten, und dass keine Luftblasen geben Sie die Schläuche. </li><li> Nach 5 min Inkubation stoppendie Pumpe und übertragen Sie den Schlauch an Leber Digest Medium. Starten Sie die Pumpe und Perfusion der Leber für eine weitere 10 bis 15 min (bis das Medium erschöpft ist). </li><li> Am Ende der Durchblutung, die Pumpe stoppen. Die Leber ist ein rosa Farbton haben und scheinen etwas vergrößert. Auszuschneiden die Leber durch sorgfältige Dissektion. Entfernen Sie die Gallenblase und übertragen die Leber zu 10 cm Gewebekulturschale. </li><li> In einer Biosicherheitswerkbank, 10 ml Plating Medium (Tabelle 1) in die Leber. Kratzen Sie vorsichtig die Leber unter Verwendung entweder einer Pinzette oder einem Skalpell, um die Leberzellen zu entfernen. Filtern der Suspension unter Verwendung eines 100 &amp; mgr; m Zellsieb und in ein konisches 50 ml-Röhrchen. Waschen Sie die Platte mit weiteren 10 ml Plattierungsmedium und Pool in der 50 ml konischen Röhrchen. </li><li> Pelletieren Sie die Zellen durch Zentrifugieren für 5 min bei 350 × g, 4 ° C. </li><li> Saugen Sie mittel- und Pellet in 10 ml Plattierungsmedium und 10 ml 90% kolloidaler Kieselsäure mit po beschichtetlyvinylpyrrolidone. Vorsichtig mischen und Zentrifuge wie in Schritt 2.11. Nach der Zentrifugation wird eine Schicht aus toten Zellen werden auf der Oberseite der Mischung schwimmt, während die lebenden Zellen auf den Grund zu pelletieren. </li><li> Saugen Sie abgestorbenen Zellen und Medium. Mit 20 ml Plattierungsmedium, Zentrifuge 2.11 waschen 2 mal. </li><li> Die Zellen in 10 ml Medium Überzug. Zählen von Zellen mit einem Hämozytometer und legen 9 x 10 4 Zellen / Vertiefung in Kollagen behandelten 24-Well-Kulturschalen. Inkubieren in einem 37 ° C Brutschrank für 2 Std. Jede Platte kann entweder für die Fettsäure-Oxidation Assay oder der Lipogenese-Assay verwendet werden. </li><li> Optional Brunnen verändern Wartung Medium (Tabelle 1) und die Kultur bei 37 ° C. 3 Tage, ohne die Testergebnisse - Zellen für 2 kultiviert werden. </li></ol> 3. Fettsäure-Oxidationsassay Warnung: Verwenden von Radioaktivität kann gefährlich sein. Alle Einkauf, Lagerung, Handhabung, und diEntsorgungsbetrieb von radioaktivem Material sollte in Übereinstimmung mit den institutionellen, staatlichen und bundesstaatlichen Vorschriften und Richtlinien ausgeführt werden. <ol><li> 16 bis 20 h vor dem Assay, waschen Zellen 2 mal mit warmem PBS. Ändern die Zellen an serumfreies Serumentzugs-Medium (Tabelle 1) mit 20 nM Glucagon und Inkubation über Nacht bei 37 ° C. Anmerkung: Da fötales Rinderserum enthält eine unbekannte Konzentration von Stoffwechsel Hormone (zB Insulin, Glukagon), Serum hungern die Zellen keine Verwechslung sich daraus für den Assay haben kann. Zellen lebensfähig in Serumhungermedium für&gt; 24 h. Glucagon Behandlung wird verwendet, um die Oxidation von Fettsäuren in den Leberzellen zu stimulieren. </li><li> Am Morgen des Tests vorzubereiten Vorinkubationsmedium: Resuspendieren eine entsprechende Menge an Natriumpalmitat in ultrareinem Wasser, um eine 100 mM Lösung und Wärme bis 70 ° C für 10 min zu machen. In der Zwischenzeit bereiten die erforderliche Menge (0,5 ml pro Vertiefung einer 24-Well-Platte)DMEM mit 25 mM HEPES, 1% BSA Fraktion V, und 20 nM Glucagon. Wärme auf 37 ° C. <ol><li> Einmal solubilisiert hinzuzufügen Palmitate auf eine Endkonzentration von 250 &amp; mgr; M zu dem Medium. Ändern Hepatozyten zu Vorinkubationsmedium und bei 37 ° C für 2 Stunden. Behalten einige Vorinkubationsmedium bei 37 ° C für die spätere Verwendung. </li></ol></li><li> Während der Inkubation Trocknen eine geeignete Menge an 14 C-Palmitat (0,5 uCi / Vertiefung) durch Verdampfung unter Stickstoff. <ol><li> Zum Beispiel, um 24 Proben in einem 24-Well-Platte zu messen, Transfer 120 ul 0,1 &amp; mgr; Ci / ml 14 C-Palmitate in ein 1,5-ml-Tube. Langsam verdampft das Ethanol-Lösungsmittel durch Einblasen von Stickstoffgas über die Lösung in einem Abstand von 3-5 cm in einem Abzug. Das Lösungsmittel sollte in etwa 30 verdampfen - 40 min, wobei Trocken 14 C-Palmitat im Boden des Röhrchens. </li></ol></li><li> Ca. 15 min vor dem Ende der Inkubation resuspendieren 14C-Palmitate in 0,1 N NaOH (12,5 ul / &amp; mgr; Ci). Inkubieren bei 70 ° C für 10 min. In drei Bänden der warmen Vorinkubationsmedium und mischen durch Auf-und Abpipettieren. </li><li> Spike jede Vertiefung mit 25 ul verdünnt 14 C-Palmitate. Mischen Sie durch vorsichtiges Schwenken der Platte und bei 37 ° C für 90 min. Dies ist die Testplatte. </li><li> Während der Inkubation, bereiten Sie die Filter Pappteller (Abbildung 1). <ol><li> Entfernen Sie die Abdeckung aus einer sterilen 24-Well-Platte. Platzieren einer 2 cm x 2 cm großes Stück Filterpapier in den Boden einer jeden der Vertiefungen. Überlagern die Platte mit einem Stück von 4 &quot;x 7&quot; Parafilm. </li><li> Mit einem großen, rechteckigen Gegenstand, beispielsweise eine Mikropipettenspitze Box, reiben Sie die Parafilm auf den Brunnen, um den Parafilm an den gut Öffnungen perforiert und eine Abdichtung zu schaffen über den Rest der Platte. Entfernen Sie die Lochkreise Parafilm jetzt Abdecken der Brunnen. Die Platte sollte jetzt fest mit Parafilm in allen Bereichen abgedeckt werden außer dem gut openings. </li></ol></li><li> 10 min vor dem Ende der Inkubation werden 200 ul 3 N NaOH zu jeder Vertiefung der Filterpapierplatte, so dass das Filterpapier absorbiert die gesamte Flüssigkeit. <ol><li> Gegebenenfalls vor dem Einfrieren, Transfer des Mediums auf eine frische 24-Well-Platte. Nach einmaligem Waschen mit PBS, lysieren die restlichen Zellen in 0,1 N HCl und berechnen Proteingehalt durch BCA-Assay. </li></ol></li><li> Am Ende der Inkubation Schnappgefrierassayplatte in flüssigem Stickstoff. Seien Sie vorsichtig, um jede Vertiefung zu gewährleisten ist komplett eingefroren, bevor Sie fortfahren. </li><li> 100 l 70% iger Perchlorsäure in jede Vertiefung der Testplatte. Unmittelbar decken mit dem Filter Pappteller. Legen Sie die Platten auf einem Orbitalschüttler und Felsen am Umlaufgeschwindigkeit von 80 rpm bei RT für 2 h. </li><li> Nach der Inkubation der Proben zu verarbeiten: <ol><li> Um den CO 2 -Anteil zu messen, übertragen Sie die Filterpapier Quadrate, um 4 ml flüssige Szintillationsflüssigkeit in einem SzintillationszählerFläschchen und Maßnahme 14 C-Signal. </li><li> Um den säurelöslichen Materials zu messen, zu übertragen 400 ul Medium in ein 1,5 ml Mikrozentrifugenröhrchen. Zentrifuge bei maximaler Geschwindigkeit für 10 min. 100 l des resultierenden Überstandes auf 500 ul von 2: 1 Chloroform-Methanol (v / v), und kurz vortexen. <ol><li> Fügen Sie 250 ul Wasser zu der Mischung und Wirbel erneut. Zentrifuge Proben für 10 min bei 3.000 x g. Transfer 200 ul der oberen Phase auf 4 ml flüssige Scintillationsflüssigkeit in einem Szintillationsfläschchen und messen 14 C-Signal. </li></ol></li></ol></li></ol> 4. Lipogenese-Assay <ol><li> Der Abend vor dem Test, waschen Sie die Zellen 2 mal mit warmem PBS. Ändern Sie die Hepatozyten, um Serumhungermedium mit 100 nM Insulin. Inkubieren über Nacht bei 37 ° C. Anmerkung: Da fötales Rinderserum enthält eine unbekannte Konzentration von Stoffwechsel Hormone (zB Insulin, Glucagon), Zu verhungern Serum die Zellen keine verwirrende Auswirkungen dies auf den Assay haben können, zu entfernen. Zellen lebensfähig in Serumhungermedium für&gt; 24 h. Insulin wird in diesem Test verwendet werden, um die Lipogenese zu stimulieren. </li><li> Machen Lipogenese Medium: Serumhungermedium mit 100 nM Insulin, 10 &amp; mgr; M kaltem Acetat und 0,5 &amp; mgr; Ci 3 H-Acetat pro Vertiefung. Wechselzellen Lipogenese Medium und bei 37 ° C für 2 Std. Fügen Sie alle Verbindungen von Interesse getestet werden. </li><li> Nach der Inkubationszeit, waschen Zellen 2 mal mit PBS. Lysieren Zellen durch Abschaben in 120 ul 0,1 N HCl. Reserve 10 &amp; mgr; l für die Protein-Assay (Beurteilung durch BCA-Test), und Transfer 100 ul bis 1,5 ml Mikrozentrifugenröhrchen. </li><li> Extrakt Lipide durch Zugabe von 500 ul von 2: 1 Chloroform-Methanol (v / v). Vortex kurz und Inkubation bei Raumtemperatur für 5 min. Fügen Sie 250 ul Wasser, Wirbel und Inkubation bei Raumtemperatur für weitere 5 min. Centrifuge Proben 10 min bei 3.000 xg, RT. Sorgsamübertragen untere Phase bis 4 ml flüssige Szintillationsflüssigkeit in einem Szintillationsfläschchen und messen 3 H-Aktivität. </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.

Die Zeit vom Opfer Perfusion sollte weniger als 3 min für ideale Perfusion und Kollagenaseverdau der Leber. Sobald Perfusion mit Perfusionsmedium eingeleitet wird, sollte die Leber sofort Aussehen von rot bis hell ändern. Nach ca. 10 min Inkubation mit LDM, wird die Leber geschwollen und rosa angezeigt. Im Falle, dass die Perfusion unzureichend ist, kann die Leber nicht zeigen diese Änderungen, und dies wird in der Regel in einem unteren Hepatozyten Ausbeute führen. Nach den Waschschritt...

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.

Hepatozyten-Isolierungen führen in der Regel in 1-3 x 10 7 Zellen insgesamt. Nach Inkubation über Nacht werden die Zellen hexagonal, von denen viele binukleären werden (Figur 2) angezeigt werden. Gesunde Zellen sollten frei von Granulationen oder Blasen, was auf Zelltod sein. Im allgemeinen wird die Oxidation von Fettsäuren Assays in drei bis vier Wiederholungen pro Testverbindung ausgeführt. Zählungen für die CO 2 Proben sind etwa ein Fünftel d...

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

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

De novo Lipogenese und β-Oxidation von Fettsäuren bilden Schlüsselstoffwechselwege in Hepatozyten, Wege, die in mehreren Stoffwechselstörungen, einschließlich Fettleber gestört werden. Hier zeigen wir Isolierung von Maus primären Hepatozyten und zu beschreiben, die Quantifizierung der β-Oxidation von Fettsäuren und Lipogenese.

Bestimmung der Fettsäureoxidation und Lipogenese in Maus primären Hepatozyten

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 Aufrufe.  University of Massachusetts Medical School.  De novo Lipogenese und β-Oxidation von Fettsäuren bilden Schlüsselstoffwechselwege in Hepatozyten, Wege, die in mehreren Stoffwechselstörungen, einschließlich Fettleber gestört werden. Hier zeigen wir Isolierung von Maus primären Hepatozyten und zu beschreiben, die Quantifizierung der β-Oxidation von Fettsäuren und Lipogenese. 

Serie: 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.

Quantitative Phosphoproteomics in Fatty Acid Stimulated 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 ...

Forschung

Biologie

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-basierte Erkennung von Aldononitrile Acetat Derivatisierte Glucosamin und Muraminsäure für Mikrobielle Rückstandsbestimmung im Boden

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%.

Isolierung und Primärkultur von Ratten Leberzellen

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

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Nahrungsergänzung von mehrfach ungesättigten Fettsäuren<em&gt; Caenorhabditis elegans</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

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;

Anwendung von Retinsäure zu Osteozyten Kulturen aus primären Maus-Osteoblasten erhalten

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

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolation und Primärkultur von Maus Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

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.

Protokoll für die Isolierung von primären humanen Hepatozyten und entsprechende Hauptpopulationen von Nicht-parenchymale Leberzellen

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.

Methoden zur Bestimmung von Preisen von Glucose und die Oxidation von Fettsäuren in der isolierten Arbeitsrattenherz

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.

Isolierung und Kultur der primären Maus Keratinozyten aus neonatalen und erwachsenen Maus Haut

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.

Isolation und 3D Kollagen Sandwich Kultur der primären Maus Hepatozyten, um die Rolle des Zytoskeletts in Bile Canalicular Formation In Vitro zu studieren

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 ...