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).

Tüm hayvan deneyleri yerel ve federal düzenlemelere uygun ve kurumsal IACUC ve radyasyon güvenliği yönetiminin onayı ile yapılmalıdır. 1. Hazırlık <ol><li> Birkaç gün önce tahlil için, Karaciğer Digest Medium (LDM) 500 ml şişe çözülme ve 50 ml konik tüpler içinde ~ 35 ml alikotları dondurursam. Kadar gerekli -20 ° C&#39;de saklayın. </li><li> Bir gün önce tahlil için, otoklav ile temiz diseksiyon araçları önceden sterilize edin. </li><li> Analiz gününde, kollajen ile 24 oyuklu kültür plakaları gerekli miktarda muamele. <ol><li> PBS ile 50 kısım, 3 mg / ml fare kuyruk kolajeni 1 kısım karıştırın. Bir 24-yuvalı plakanın her oyuğuna yaklaşık 500 ul ilave edin ve oda sıcaklığında 3-5 dakika boyunca (RT) enkübe edilir. Kollajen çıkarın ve plaka kaput kuru hava için izin verir. En az bir ek tekrarlayın. Not: Kollajen kaplama reklamda bir haftaya kadar yapılabilirvance ve plakalar plastik sargı mühürlü 4 ° C&#39;de saklandı. </li></ol></li><li> 37 ° C&#39;ye kadar çözülme LDM ve sıcak ve 1 N KOH kullanılarak 7.4&#39;e pH ayarlamak: deney için LDM hazırlayın. Bir çevrimli 42 ° C su banyosunda 0.2 mikron şırınga filtresi ve yer kullanarak Filtre. </li><li> Su banyosu çevrimli 42 ° C&#39;ye kadar Karaciğer Perfüzyon Orta Sıcak 25 mi. </li><li> Polivinilpirolidon çözeltisi ile kaplanmış% 90 kolloidal silika hazırlayın: polivinilpirrolidon ile kaplanmış koloidal silika 9 ml 10x DPBS 1 ml ilave edilir. 0.1 N HCI kullanılarak pH 7.4&#39;e ayarlayın. Steril 0.2 um şırınga filtresi ile filtre. Oda sıcaklığında saklayın. </li><li> 37 ° C sıcak bir orta kaplama. </li></ol> İlköğretim Fare Hepatositlerin 2. izolasyonu <ol><li> Peristaltik pompa, boru ve diseksiyon tablo ayarlama: steril su, 10 ml, ardından damıtılmış su içinde 5 mi,% 70 etanol ile yıkama ile boru sterilize edin. Doldurmak için Karaciğer Perfüzyon Ortamında boru ve koşmak pompayı yerleştirinborunun tüm uzunluğu. </li><li> Kurumsal onaylı yöntem ile fare Kurban. </li><li> Karın boşluğuna açık parçalara ayır:% 70 etanol ile liberal fare karın püskürtün. Künt uç makas kullanarak, dermis yoluyla karın uzunluğunu orta hat kesi yapmak ve yanal yansıtır. Iç organları maruz periton benzer bir kesi yapmak. <ol><li> Yavaşça bağırsak yerinden, künt aleti kullanarak karın damarlanma karın inferior vena kava (IVC) bulun ve renal ven distal damar altına bir dikiş, koyun ortaya çıkarmak için </li></ol></li><li> Distal sütür için, dikiş düzeyde ötesine ilerleyen, IVC içine bir iğne ve kateter yerleştirin. Hala yerinde iğne ile yerde tutmak için kateter etrafında dikiş kravat. Dikkatlice iğneyi çıkartın. Eğer kateter yoluyla akışı ile, doğru bir şekilde kan yapılır. </li><li> Bir pipet kullanarak, sağlanması, perfüzyon ortamına kateter kalan alanı doldurmakBu hava mevcut. Büyük bir özenle, kateter tüp takmak. </li><li> Akciğer boşluğuna açık parçalara ayır: hafifçe diyaframa maruz üstün karaciğer lobları yansıtmaktadır. Dikkatle sonra keskin uçlu makas ile diyafram delinme safra kesesi ve plevra damarsal önlemek için özen, plevra boşluğu maruz yanal kesi yapmak. Hepatik ven sadece proksimal torasik inferior vena kava etrafında bir bulldog kelepçe yerleştirin. </li><li> Portal ven kesin ve 3 pompa açmak - / dk 4 ml. Perfüzyon ortamı, yaklaşık 20 ml ile 5 dakika boyunca karaciğer Perfüzyon Ortamı ile karaciğer serpmek. Not: Karaciğer hemen tan / gri kırmızı değişmelidir. Karaciğer bölümleri kırmızı kalırsa, bu büyük olasılıkla kötü perfüzyon gösterir ve başarılı izolasyon olasılığı büyük ölçüde azalır. Perfüzyon sırasında dışarı çalışmaz ortamı sağlamak için dikkatli olun ve hiçbir kabarcıklar tüp girmek söyledi. </li><li> 5 dakika inkübasyondan sonra, stopPompa ve Karaciğer Digest Orta boru aktarın. (Orta tükenene kadar) 15 dakika - pompayı yeniden başlatın ve başka bir 10 için karaciğer serpmek. </li><li> Perfüzyon sonunda pompayı durdurun. Karaciğer pembemsi renge sahip ve biraz genişlemiş görünmelidir. Dikkatli diseksiyon ile karaciğer tüketim. Safra kesesi çıkarın ve 10 cm doku kültürü çanak karaciğer aktarmak. </li><li> Bir biyogüvenlik kabini, karaciğere Kaplama Orta 10 ml (Tablo 1) ekleyin. Yavaşça hepatositler kaldırmak ya forseps veya neşter kullanılarak karaciğer kazıyın. 50 ml&#39;lik konik tüp 100 mikron hücre süzgeç ve transfer kullanarak süspansiyon Filtre. 50 ml konik tüp ek bir 10 Kaplama Orta ml ve havuzlu plaka yıkayın. </li><li> 350 xg, 4 ° C&#39;de 5 dakika boyunca santrifüj hücreleri Pelet. </li><li> % 90 koloidal silika, 10 mi po ile kaplanmış Kültür ortamı basınçla Kaplama Orta 10 ml tekrar süspansiyon pelet ekleyinlyvinylpyrrolidone. Adım 2.11 gibi yavaşça ve santrifüj karıştırın. Canlı hücreler, alt pelet ise santrifüjlemeden sonra, ölü hücreleri bir tabakası, karışımın üzerinde yüzen olacaktır. </li><li> Aspire ölü hücreler ve orta. Kaplama Medium, 2.11 gibi santrifüj 20 ml 2 kez yıkayın. </li><li> Kaplama Orta 10 ml süspanse hücreleri. Bir hemasitometre ile hücre sayımı ve kollajen ile tedavi edilmiş 24 oyuklu kültür tabaklarında 9 x 10 4 hücre / çukur yerleştirin. 2 saat boyunca 37 ° C&#39;lik bir doku kültürü kuluçka makinesi içinde inkübe edin. Her plaka, yağ asidi oksidasyon deneyi TD lipogenesis tahlilinde biri için kullanılabilir. </li><li> İsteğe bağlı olarak, 37 ° C &#39;de, muhafaza edici ortam (Tablo 1) ve kültür kuyularının değiştirin. Deney sonuçları etkilemeden 3 gün - Hücreler 2 kültürlenebilir. </li></ol> 3. Yağ Asidi Oksidasyonu Deneyi Uyarı: radyoaktivite kullanılması tehlikeli olabilir. Tüm satın alma, depolama, elleçleme, ve diRadyoaktif maddenin sposal kurumsal, eyalet ve federal düzenlemeler ve kurallara uygun olarak yapılmalıdır. <ol><li> 16 - 20 saat önce tahlil için, ılık PBS ile hücreler 2 kez yıkayın. 20 nM glukagon ile serumsuz serum açlık Ortamı (Tablo 1) hücreleri değiştirme ve gece boyunca 37 ° C&#39;de inkübe edin. Not: fetal sığır serumu metabolik hormonların bilinmeyen bir konsantrasyon (örneğin insülin, glukagon) içerdiğinden, serum bu tahlilde üzerinde sahip olabileceği herhangi karıştırıcı etkileri ortadan kaldırmak için hücreleri açlıktan. Hücreler&gt; 24 saat serum Açlık Ortamda yaşayabilir. Glukagon işleme hepatositlerde yağlı asit oksidasyonunu teşvik etmek için kullanılır. </li><li> Tahlilinde sabahı, ön inkübasyon ortamı, hazırlanması: 10 dakika boyunca 70 ° C&#39;ye kadar, bir 100 mM çözelti ve ısı yapmak için, aşırı saf su içinde sodyum-palmitat, uygun bir miktarda yeniden süspanse edin. Bu arada, gerekli miktarda (24-iyi levha başına 0.5 mi) hazırlanması25 mM HEPES,% 1 BSA fraksiyonu V, ve 20 nM glukagon ile DMEM. 37 ° C&#39;ye ısıtın. <ol><li> Çözündürülmüş sonra, ortama 250 uM nihai konsantrasyona kadar palmitat ekleyin. Orta-Kuluçka öncesi ve 2 saat 37 ° C&#39;de inkübe hepatositlerin değiştirin. Daha sonra kullanılmak üzere 37 ° C &#39;de, bazı ön inkübasyon ortamı, ayırın. </li></ol></li><li> İnkübasyon sırasında, nitrojen gazı altında buharlaştırma ile 14 C-palmitat, uygun bir miktarda (0,5 uCi / oyuk) kurutun. <ol><li> Örneğin, bir 24-çukurlu plaka içinde 24 örnekleri ölçmek için 1,5 ml&#39;lik bir tüpe 0.1 uCi / ml 14 C-palmitat 120 ul transfer. Yavaş yavaş bir duman başlığı içinde 3-5 cm uzaklıktan çözelti üzerinden bir nitrojen gazı püskürtülerek solvent etanol buharlaştırılır. Tüpün alt kuru 14C-Palmitat bırakarak, 40 dakika - çözücü içinde yaklaşık 30 buharlaşır gerekir. </li></ol></li><li> Yaklaşık 15 dakika süreyle inkübe edildikten sona ermeden önce, 14 tekrar süspansiyon0,1 N NaOH içerisinde C = Palmitat (12.5 ul / uCi). 10 dakika boyunca 70 ° C&#39;de inkübe edin. Sıcak ön inkübasyon ortamı, üç hacim ekleyin ve aşağı yukarı pipetleme karıştırılır. </li><li> Seyreltilmiş 14 C-palmitat 25 ul her bir başak. Hafifçe plaka sallanmasıyla karıştırın ve 90 dakika boyunca 37 ° C&#39;de inkübe edin. Bu deney, bir plakadır. </li><li> İnkübasyon sırasında, Filtre Kağıdı Plate (Şekil 1) hazırlar. <ol><li> Steril 24 plaka kapağı çıkarın. Kuyucuğun alt filtre kağıdı, bir 2 cm x 2 cm parçası yerleştirin. 4 &quot;x 7&quot; parafilm bir parça ile plaka Yerleşimi. </li><li> Böyle bir mikropipet ucu kutusu gibi, büyük dikdörtgen bir nesne kullanarak, iyi açılışlarında parafilm perfore ve plaka geri kalanı üzerinde bir mühür oluşturmak için kuyular üzerinde parafilm ovalayın. Şimdi kuyuları kapsayan parafilm delikli çevreleri çıkarın. Plaka şimdi sıkı sıra o hariç tüm alanlarda parafilm ile kaplanmış olmalıdırpenings. </li></ol></li><li> 10 dakika süreyle inkübe edildikten sona ermeden önce, bir filtre kağıdı tüm sıvı absorbe emin olarak, filtre kağıdı bir levhanın her çukuruna 3 N NaOH 200 ul ilave edin. <ol><li> Donmaya karşı isteğe bağlı olarak önceden bir taze 24-yuvalı plakaya orta aktarın. Bir kez PBS ile yıkandıktan sonra, 0.1 N HCI kalan hücreler lize edildi ve bir BCA aracıyla protein içeriğini hesaplar. </li></ol></li><li> İnkübasyonun sonunda, sıvı nitrojen içinde Deney Plakası ek dondurularak. Tamamen geçmeden önce dondurulur her iyi sağlamak için dikkatli olun. </li><li> Deney plakası, her sıra için% 70 perklorik asit, 100 ul ekle. Hemen Filtre Kağıt Plate ile kaplayın. 2 st için oda sıcaklığında 80 rpm&#39;de yörüngesel hızda bir orbital çalkalayıcı ve kaya plakaları yerleştirin. </li><li> İnkübasyondan sonra, örnekler işlem: <ol><li> Bir sintilasyon 4 ml sıvı sintilasyon sıvısına filtre kağıdı kareler transfer CO 2 fraksiyonu ölçmek içinŞişe ve ölçü 14 C sinyali. </li><li> Asit çözünebilir malzeme ölçen 1.5 ml mikrofüj tüpe orta 400 ul aktarma. 10 dakika süreyle maksimum hızda santrifüjleyin. Kısaca 1 kloroform-metanol (h / h) ve girdap: 2 500 ul Elde edilen üstte yüzere, 100 ul ekle. <ol><li> Yine karışımına su 250 ul ekleyin ve vorteks. 3,000 x g&#39;de 10 dakika boyunca santrifüj örnekleri. Transfer sintilasyon şişesine 4 ml sıvı sintilasyon sıvısı üst fazın 200 ul 14 C sinyali ölçer. </li></ol></li></ol></li></ol> 4. lipogenez Deneyi <ol><li> Akşam önce tahlil başlamadan, ılık PBS ile hücreler 2 kez yıkayın. 100 nM insülin Serum Açlık Orta hepatositler değiştirin. 37 ° C&#39;de gece boyunca inkübe edin. Not: fetal sığır serumu bir metabolik hormonların bilinmeyen konsantrasyonu (örneğin insülin içerdiğinden, glukagon), Serum Bu testte üzerinde sahip olabileceği herhangi karıştırıcı etkileri ortadan kaldırmak için hücreleri açlıktan. Hücreler&gt; 24 saat serum Açlık Ortamda yaşayabilir. İnsülin lipogenez uyarmak için bu deneyde kullanılır. </li><li> 100 nM insülin, 10 uM soğuk asetat ve 0.5 uCi 3H-asetat başına iyi Serum Açlık Orta: lipogenezi Orta olun. Lipogenez Ortama hücreleri değiştirmek ve 2 saat süre ile 37 ° C&#39;de inkübe edin. Çıkar bileşiklerin, test edilecek içerir. </li><li> İnkübasyon süresinden sonra, hücreler PBS ile 2 kez yıkayın. 0.1 N HCI içinde 120 ul kazınarak Hücreleri,. Rezerv 10 protein tayini için ul (BCA tahlili ile değerlendirilmesi), ve transfer 1,5 ml mikrofüj tüpüne 100 ul. </li><li> 2 500 ul ilave edilerek özü lipidler: 1 kloroform-metanol (h / h). Vorteks kısa ve 5 dakika boyunca oda sıcaklığında inkübe edilir. Su, girdap 250 ul ekleyin ve ek 5 dakika boyunca oda sıcaklığında inkübe edilir. Santrifüj örnekleri 3000 x g&#39;de oda sıcaklığında 10 dakika. ÖzenleBir sintilasyon şişesine 4 ml sıvı sintilasyon sıvısına alt faz transferi ve 3 H aktivitesini ölçmek. </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.

Perfüzyon kurban gelen zaman ideal bir perfüzyon ve karaciğer kollajenaz sindirimi için en az 3 dakika olmalıdır. Perfüzyon Orta ile perfüzyon başlatılır sonra, karaciğer hemen kırmızı soluk ila görünümünü değiştirmek gerekir. LDM inkübasyonu yaklaşık 10 dakika sonra, karaciğer, şişmiş ve pembe görünür. Perfüzyon yetersiz olması halinde, karaciğer, bu değişiklikler göstermezler olabilir ve bu, tipik olarak daha düşük bir hepatosit verimle sonuçlanır. <p class="jove_content"...

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.

3 x 10 7 toplam hücre - hepatosıt izolasyonlar genellikle 1 ile sonuçlanır. Gece boyunca inkübasyondan sonra, hücreler (Şekil 2), binükleer olacak birçoğu altıgen görünür. Sağlıklı hücreler, hücre ölümü göstergesidir granülasyon veya kabarcıkların, yoksun olmalıdır. Genel olarak, yağ asidi oksidasyon deneyi, test bileşiği başına üç ila dört kez tekrarlanmış çalıştırılır. CO2 numune için sayımlar asit çözü...

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 lipogenez ve β-yağ asidi oksidasyon hepatosit kilit metabolik, yağlı karaciğer hastalığı da dahil olmak üzere birçok metabolik bozukluklar, içinde tedirgin olan yollar oluşturmaktadır. Burada fare primer hepatositlerin izolasyonu göstermek ve β-yağ asidi oksidasyon ve lipogenez ölçümü açıklar.

Fare İlköğretim Hepatositlerde Yağ Asidi Oksidasyon ve lipogenez Belirlenmesi

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 Görüntüleme.  University of Massachusetts Medical School.  De novo lipogenez ve β-yağ asidi oksidasyon hepatosit kilit metabolik, yağlı karaciğer hastalığı da dahil olmak üzere birçok metabolik bozukluklar, içinde tedirgin olan yollar oluşturmaktadır. Burada fare primer hepatositlerin izolasyonu göstermek ve β-yağ asidi oksidasyon ve lipogenez ölçümü açıklar. 

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.

Yağ Asidi Kantitatif Phosphoproteomics Uyarılmış 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 ...

Biyoloji

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.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

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.

Toprak Mikrobiyal Kalıntı Tayini için Aldononitrile Asetat türevlendirilmiş Glukozamin ve Muramic Asit GC tabanlı Algılama

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%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

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;

İlköğretim Fare Osteoblastlar gelen osteositlere Kültürler Edinme Retinoik Asit Uygulama

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.

İzolasyon ve Fare aort endotel hücreleri İlköğretim Kültür

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.

İlköğretim İnsan Hepatositlerin İzolasyonu ve Non-parankimal karaciğer hücrelerinin Binbaşı popülasyonları İlgili Protokolü

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.

İzole Çalışma Sıçan Kalp Glukoz ve Yağ Asidi Oksidasyonu Oranlarının Belirlenmesine Yöntemleri

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.

Yenidoğan ve Erişkin Fare Ciltlerindeki Primer Fare Keratinositlerin İzolasyonu ve Kültürü

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.

İzolasyon ve 3D Kollajen Sandviç Kültürü Birincil Fare Hepatositler Safra Canaliküler Oluşumunda Sitoskeleton Rolünü Çalışma 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 ...