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).

すべての動物実験は、ローカルおよび連邦規制に従って及び制度IACUCと放射線安全管理の承認を得て実施されるべきです。 1.準備<ol><li>数日前、アッセイに、肝臓ダイジェストミディアム（LDM）の500ミリリットル瓶を解凍し、50mlのコニカルチューブに〜35ミリリットルのアリコートを再凍結。必要になるまで-20℃で保存してください。 </li><li>アッセイの1日前に、オートクレーブによりクリーンな解剖ツールを事前に滅菌します。 </li><li>アッセイの日に、コラーゲンとの24ウェル培養プレートの必要な量を扱います。 <ol><li> PBSの50部で3 mg / mlのラット尾コラーゲン1部を混合します。 24ウェルプレートの各ウェルに約500μLを加え、室温（RT）で3~5分間インキュベートします。コラーゲンを外し、プレートをフード内空気乾燥することができます。少なくとも一つの追加の時間を繰り返します。 注意：コラーゲンコーティングが広告に1週間まで行うことができますヴァンス、プレートをプラスチックラップで密封し、4℃で保存しました。 </li></ol></li><li> 37℃に解凍LDMと暖かいと1N KOHを使用してpHを7.4に調整します。アッセイのLDMを準備します。再循環を42℃の水浴中で、0.2μmのシリンジフィルターと場所を使用してフィルタ。 </li><li>水浴を再循環で42°Cまでの肝灌流中のウォーム25ミリリットル。 </li><li>ポリビニルピロリドンの溶液を塗布し、90％のコロイダルシリカを準備します。ポリビニルピロリドンで被覆されたコロイド状シリカの9ミリリットルに10倍のDPBS 1ミリリットルを追加します。 0.1 N HClを用いてpHを7.4に調整します。滅菌0.2μmのシリンジフィルターでろ過します。室温で保管してください。 </li><li> 37°Cまでのめっき中暖かいです。 </li></ol>初代マウス肝細胞の単離2。 <ol><li>蠕動ポンプ、チューブ、および解剖テーブルを設定します。滅菌水10ml、続いて蒸留水で5ミリリットルの70％エタノールで洗浄することによりチューブを滅菌します。埋めるために肝灌流培地でチューブと実行ポンプを配置しますチューブの全長。 </li><li>制度承認の方法により、マウスを生け贄に捧げます。 </li><li>腹腔を開き、解剖70％エタノールで自由にマウスの腹部スプレー。平滑末端はさみを使用して、真皮を通って腹部の長さを正中切開を行い、横方向に反映しています。内臓を露出するために腹膜に同様の切開を行います。 <ol><li>鈍器を使用して、静かに腎静脈の遠位に、腹部下大静脈（IVC）を見つけて、血管の下に縫合糸を配置腹部血管系を露出するように腸を変位</li></ol></li><li>遠位縫合糸に、縫合糸のレベルを超えて、それを進める、IVCに針やカテーテルを配置します。まだ場所に針で、所定の位置に保持するために、カテーテルの周りに縫合糸を結びます。慎重に針を取り除きます。カテーテルを通して流れに血液、正しく行われた場合。 </li><li>ピペットを使用することで、確実に、灌流媒体とカテーテル内の残りの領域を埋めます空気が存在しません。細心の注意を払って、カテーテルにチューブを取り付けます。 </li><li>肺の空洞を開く解剖：優しくダイヤフラムを露出するために、優れた肝葉を反映しています。慎重に胆嚢と胸膜血管系を避けるように注意しながら、胸膜腔を露出するために、横方向の切開を行い、その後、鋭い先端のハサミで隔膜を穿刺。肝静脈のすぐ近位胸部下大静脈の周りブルドッグクランプを配置します。 </li><li>門脈をカットし、3でポンプの電源をオンに - 4ミリリットル/分。灌流培地の約20ミリリットルを用いて5分間肝灌流培地で肝臓を灌流。 注：肝臓はすぐにグレー/黄褐色に赤から変更する必要があります。肝臓の部分が赤のままである場合、これはおそらく乏しい灌流を示し、成功した分離の可能性が大幅に減少します。灌流の間に、媒体が出て実行されず、気泡がチューブに入らないことを保証するように注意してください。 </li><li> 5分間のインキュベーションに続いて、停止しますポンプおよび肝臓ダイジェスト中にチューブを移します。 （媒体がなくなるまで）15分 - ポンプを再起動して、別の10のために肝臓を灌流。 </li><li>灌流の終了時に、ポンプを停止します。肝臓はピンクがかった色合いを持っており、やや拡大表示されます。慎重な切開により、肝臓を切除。胆嚢を削除し、10cmの組織培養皿に肝臓を転送します。 </li><li>バイオセーフティキャビネットでは、肝臓にメッキミディアム（ 表1）の10ミリリットルを追加します。静かに肝細胞を除去するために、いずれかのピンセットやメスを用いて肝臓をこすり。 50mlコニカルチューブに100μmのセルストレーナー、転送を使用してサスペンションをフィルタリングします。 50mlコニカルチューブにめっき中、プールの追加10mlでプレートを洗浄します。 </li><li> 350×gで、4℃で5分間の遠心分離により細胞をペレット化。 </li><li>めっき中の10ml中の培地を吸引し、再懸濁ペレットとPOでコーティングされた90％のコロイダルシリカの10ミリリットルを追加しますlyvinylpyrrolidone。穏やかに混合し、ステップ2.11のように遠心します。生細胞が底にペレット化する一方遠心分離した後、死細胞の層は、混合物の上に浮遊されます。 </li><li>吸引し死んだ細胞および培地。メッキ中、2.11のように遠心20mlで2回洗浄します。 </li><li>めっき中の10ml中に再懸濁細胞。血球計数器で細胞をカウントし、コラーゲン処理した24ウェル培養皿に9×10 4細胞/ウェルで置き。 2時間、37℃の組織培養インキュベーター中でインキュベートします。各プレートは、脂肪酸酸化アッセイまたは脂質生成アッセイのいずれかのために使用することができます。 </li><li>必要に応じて、37℃で維持培地（ 表1）と文化に井戸を変更します。アッセイの結果に影響を与えずに3日 - 細胞を2ために培養することができます。 </li></ol> 3.脂肪酸酸化アッセイ警告：放射能の使用は危険です。すべての購買、保管、取り扱い、およびジ放射性物質のsposalは、制度的状態、および連邦の規制やガイドラインに従って行われるべきです。 <ol><li> 16から20時間アッセイの前に、温かいPBSで細胞を2回洗浄します。 20 nMのグルカゴンで無血清血清飢餓培地（ 表1）に細胞を変更し、37℃で一晩インキュベートします。 注意：ウシ胎児血清は、代謝ホルモンの未知の濃度（ 例えば、インスリン 、グルカゴン）を含んでいるので、血清これはアッセイに持つかもしれない交絡の影響を除去するために、細胞を飢えさせます。細胞は&gt; 24時間血清飢餓培地中で生存可能です。グルカゴン治療は、肝細胞内の脂肪酸酸化を刺激するために使用されます。 </li><li>アッセイの朝に、前培養培地を準備し、10分間70℃で100mMの溶液と熱を加えるために超純水にナトリウムパルミチン酸の適切な量を再懸濁します。一方で、必要量（24ウェルプレートのウェルあたり0.5 ml）を調製25mMのHEPES、1％のBSAフラクションV、および20 nMのグルカゴンを含むDMEMの。 37℃に加熱します。 <ol><li>可溶化した後、培地に250μmの最終濃度にパルミチン酸を追加します。プレインキュベーション中に肝細胞を変更し、37℃で2時間インキュベートします。後で使用するために、37℃で、いくつかのプレインキュベーション中を予約。 </li></ol></li><li>インキュベーションの間、窒素ガス下で蒸発させて14 C-パルミチン酸（0.5μCiの/ウェル）の適切な量を乾燥させます。 <ol><li>例えば、24ウェルプレートで24のサンプルを測定して1.5 mlチューブに0.1μCiの/ mlの14 C-パルミチン酸120μlのを転送します。ゆっくりとヒュームフード3-5センチの距離からの溶液上に窒素ガスを吹き付けてエタノール溶媒を蒸発させます。チューブの底に乾燥14 C-パルミチン酸を残し、40分-溶媒が約30を蒸発させる必要があります。 </li></ol></li><li>約15分間のインキュベーションの終了前に、14を再懸濁0.1 N NaOH中のC-パルミチン酸（12.5μL/マイクロCi）。 70℃で10分間インキュベートします。暖かいプレインキュベーション中の三巻を追加し、ピペッティングにより混和します。 </li><li>希釈された14 C-パルミチン酸の25μlの各ウェルをスパイク。静かにプレートをロッキングすることによって混合し、37℃で90分間インキュベートします。これは、アッセイプレートです。 </li><li>インキュベーションの間、フィルターペーパープレート（ 図1）を作製 。 <ol><li>滅菌24ウェルプレートからカバーを取り外します。各ウェルの底にろ紙の2センチメートル×2センチピースを置きます。 4 &quot;×7&quot;パラフィルムの切れ端でプレートをオーバーレイ。 </li><li>このようなピペットチップボックスとして、大きな長方形のオブジェクトを使用して、ウェル開口部にパラフィルムを穿孔し、プレートの残りの部分の上にシールを作成するために、ウェル上にパラフィルムをこします。今井戸をカバーするパラフィルムの穴あき円を削除します。プレートは現在、しっかりウェルOを除くすべての地域でパラフィルムでカバーされるべきですpenings。 </li></ol></li><li>インキュベーションの終了の10分前に、ろ紙は、液体の全てを吸収し確認して、フィルターペーパープレートの各ウェルに3 N NaOHを200μlを添加します。 <ol><li>必要に応じて前に新鮮な24ウェルプレートにメディアを転送し、凍結します。 PBSで1回洗浄した後、0.1 N HCl中の残りの細胞を溶解し、BCAアッセイによってタンパク質含量を算出します。 </li></ol></li><li>インキュベーションの終了時に、スナップ凍結アッセイプレートを、液体窒素中で。各ウェルは、先に進む前に、完全に凍結されていることを確認するように注意してください。 </li><li>アッセイプレートの各ウェルに70％過塩素酸を100μlのを追加します。すぐにフィルターペーパープレートでカバーしています。室温で2時間80 rpmでの軌道速度でオービタルシェーカーと岩の上にプレートを置きます。 </li><li>インキュベーション後、サンプルを処理します。 <ol><li>シンチレーション4 mlの液体シンチレーション液をろ紙の正方形を転送する、CO 2の画分を測定します14 C信号をバイアルおよび測定します。 </li><li>酸可溶性物質を測定するために、1.5mlマイクロチューブに培地を400μlのを転送します。 10分間最大速度で遠心します。簡単に1クロロホルム - メタノール（v / v）であり、渦：2の500μlに得られた上清を100μlを追加します。 <ol><li>再び混合物に水を250μl加え、ボルテックス。 3000×gで10分間遠心サンプル。シンチレーションバイアルに液体シンチレーション流体4ミリリットルに上相の200μLを移し、14 C信号を測定します。 </li></ol></li></ol></li></ol> 4.脂質生成アッセイ<ol><li>アッセイの前に開始する夜は、温かいPBSで細胞を2回洗浄します。 100nMのインスリンで血清飢餓中に肝細胞を変更します。 37℃で一晩インキュベートします。 ウシ胎児血清は代謝ホルモンの未知の濃度が含まれているので（ 例えば、インスリン 、グルカゴン注：）、血清がこのアッセイに持つかもしれない交絡の影響を除去するために、細胞を飢えさせます。細胞は&gt; 24時間血清飢餓培地中で生存可能です。インスリンは、脂質生成を刺激するために、このアッセイで使用されます。 </li><li> 100nMのインスリン、10μMの冷たい酢酸および0.5μCiの3 H-アセテートウェル当たりで血清飢餓培地：脂質生成中にします。脂質生成中に細胞を変更し、37℃で2時間インキュベートします。試験しようとする目的の任意の化合物が含まれます。 </li><li>インキュベーション期間の後、PBSで細胞を2回洗浄します。 0.1 N HClを120μlの掻き落とし行ない、細胞を溶解します。リザーブ10タンパク質アッセイ用μL（BCAアッセイにより評価）、および1.5mlマイクロチューブに100μlのを転送します。 </li><li> 1クロロホルム - メタノール（v / v）の：2の500μlに添加することにより、脂質を抽出します。渦は簡単にし、5分間室温でインキュベートします。水、渦の250μLを加え、さらに5分間室温でインキュベートします。遠心サンプル3000×gで、室温で10分。慎重にシンチレーションバイアルに4 mlの液体シンチレーション流体に下相を転送し、3 H活性を測定します。 </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.

犠牲から灌流までの時間は、肝臓の理想的な灌流およびコラゲナーゼ消化のために3分未満でなければなりません。灌流培地で灌流が開始されると、肝臓はすぐに淡赤からに外観を変更する必要があります。 LDMとのインキュベーションの約10分後、肝臓が腫れてピンク色に表示されます。潅流が不十分である場合には、肝臓はこれらの変化を示さないことがあり、これは典型的にはより低い肝...

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.

3×10 7の全細胞-肝細胞の単離は、一般的に1になります。一晩のインキュベーションの後、細胞は、六角形の表示され、その多くは（ 図2）二核れます。健康な細胞は、細胞死の指標である顆粒またはブレブ、を欠いでなければなりません。 一般的には、脂肪酸酸化アッセイは、試験化合物ごとに3〜4回繰り返して実行されます。 CO 2のサンプ?...

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

デノボ脂質生成およびβ脂肪酸酸化は、肝細胞に脂肪肝疾患を含むいくつかの代謝障害に摂動された経路を主要な代謝経路を構成しています。ここでは、マウス初代肝細胞の単離を実証し、β - 脂肪酸の酸化および脂質生成の定量化について説明します。

マウス初代肝細胞における脂肪酸酸化および脂質生成の決意

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 ビュー.  University of Massachusetts Medical School.  デノボ脂質生成およびβ脂肪酸酸化は、肝細胞に脂肪肝疾患を含むいくつかの代謝障害に摂動された経路を主要な代謝経路を構成しています。ここでは、マウス初代肝細胞の単離を実証し、β - 脂肪酸の酸化および脂質生成の定量化について説明します。 

ビデオ: 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.

脂肪酸の定量的なPhosphoproteomicsは刺激を受けてサッカロマイセスセレビシエ</em

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

生物学

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.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

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.

土壌中の微生物の残留決定のためのAldononitrile酢酸誘導体化グルコサミンとムラミン酸のGCベースの検出

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%.

ラット肝細胞の単離及び初代培養

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;

初代マウス骨芽細胞から骨細胞培養物を入手するレチノイン酸のアプリケーション

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

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

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

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

初代ヒト肝細胞と非実質肝細胞の対応する主な集団の単離のためのプロトコル

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.

単離されたワーキングラット心臓におけるグルコースおよび脂肪酸酸化の宿泊料金を決意するための方法

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.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

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.

インビトロにおける胆道カネラ形成における細胞骨格の役割を研究する原発マウス肝細胞の単離と3Dコラーゲンサンドイッチ培養

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

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

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

多パラメータ灌流制御による原発性ラット肝細胞の単離

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