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).

모든 동물 실험은 지역 및 연방 규정에 따라 및 기관 IACUC 및 방사선 안전 관리의 승인을 수행해야한다. 1. 준비 <ol><li> 몇 일 전에 분석에, 간 다이제스트 중간 (LDM)의 500㎖의 병을 해동하고 50 ML 원뿔 튜브 ~ 35 mL를 분취을 재 냉동. 필요할 때까지 -20 ℃에서 보관하십시오. </li><li> 어느 날 이전의 분석에, 오토 클레이브에 의해 깨끗한 해부 도구를 미리 소독. </li><li> 분석 당일, 콜라겐 24- 웰 배양 플레이트의 필요량을 치료. <ol><li> PBS의 50 부를 3 ㎎ / ㎖ 래트 꼬리 콜라겐 1 부를 섞는다. 24 웰 플레이트의 각 웰에 약 500 μl를 추가하고 실온에서 3 ~ 5 분 (RT) 동안 품어. 콜라겐을 제거하고 판은 후드에서 공기 건조 할 수 있습니다. 적어도 하나의 추가 시간을 반복합니다. 참고 : 콜라겐 코팅이 광고에 일주까지 수행 할 수 있습니다밴스와 플레이트는 플라스틱 포장에 밀​​봉 4 ℃에서 보관. </li></ol></li><li> 37 ° C로 해동 LDM 및 따뜻하고 1 N KOH를 사용하여 7.4으로 산도를 조정합니다 분석을 위해 LDM을 준비합니다. 순환 42 ° C의 물을 욕조에 0.2 μm의 주사기 필터와 장소를 사용하여 필터. </li><li> 물 목욕을 순환 42 ° C까지 간 관류 중간의 따뜻한 25 ml의. </li><li> 폴리 비닐 피 롤리 돈의 용액으로 코팅 90 % 콜로 이달 실리카를 준비 : 폴리 비닐 피 롤리 돈으로 코팅 된 콜로 이달 실리카의 9 ml의 10 배 DPBS의 1 ML을 추가합니다. 0.1 N 염산을 사용하여 pH 7.4까지를 조정합니다. 멸균 0.2 μm의 주사기 필터로 필터링. 실온에서 보관하십시오. </li><li> 37 ° C의 따뜻한 도금 중간. </li></ol> 기본 마우스 간세포 2. 분리 <ol><li> 연동 펌프, 튜브 및 해부 테이블을 설정 : 멸균 물 10ml에 의해 증류수에 5 ml의 70 % 에탄올로 세척하여 튜브를 소독. 채우기 위해 간 관류 중간에 튜브 및 실행 펌프를 배치튜브의 전체 길이. </li><li> 제도적으로 승인 된 방법으로 마우스를 희생. </li><li> 복강을 오픈 해부 : 70 % 에탄올로 자유롭게 마우스 복부를 스프레이. 무딘 엔드 가위를 사용하여 진피를 통해 복부의 길이를 중간 선 절개를하고 옆으로 반영합니다. 내장을 노출 복막에 비슷한 절개를합니다. <ol><li> 부드럽게 장을 대체, 둔기를 사용하면 복부 혈관이 복부 하대 정맥 (IVC)을 찾아 신장 정맥에 말초 혈관 아래에 봉합을 배치 노출 </li></ol></li><li> 말초 봉합사로 봉합의 수준을 넘어 그것을 발전, IVC에 바늘과 카테터를 배치합니다. 아직도 그 자리에 바늘로, 제자리에 고정하기 위해 카테터 주위의 봉합을 묶어. 조심스럽게 바늘을 제거합니다. 경우는 카테터를 통해 흐름을 제대로 혈액을 수행. </li><li> 피펫을 사용하여, 보장, 관류 매체와 카테터의 나머지 영역을 채우아무 공기는 존재하지 않는다. 큰 관심과 함께, 카테터에 튜브를 연결합니다. </li><li> 폐의 구멍을 개방 해부 : 부드럽게 다이어프램을 노출 우수한 간 로브를 반영합니다. 조심스럽게 다음, 날카로운 팁 가위로 진동판에 구멍 담낭 및 흉막 혈관을 피하기 위해주의하면서, 흉강을 노출하는 측면 절개를합니다. 간정맥 단지 근위 흉부 하대 정맥 주위 불독 클램프를 놓습니다. </li><li> 포털 정맥을 잘라 3에서 펌프를 켜십시오 - / 분 4 ml의. 관류 매체의 약 20 ㎖을 사용하여 5 분 간 관류 매체 간을 관류. 참고 : 간은 즉시 갈색 / 회색 빨간색에서 변경해야합니다. 간 부를 레드 남아 있으면,이 가능성이 불량한 관류를 나타내며, 성공적인 분리의 가능성이 크게 감소된다. 관류하는 동안 밖으로 실행되지 않습니다 매체를 보장하기 위해주의와 기포가 튜브를 입력 없다. </li><li> 5 분 배양을 따르면, 중지펌프 및 간 다이제스트 중간에 튜브를 전송합니다. (매체가 소진 될 때까지) 15 분 - 펌프를 다시 시작하고 또 다른 10 간을 관류. </li><li> 관류 펌프를 멈춘다. 간은 분홍빛이 도는 색상이 다소 확대 나타납니다. 주의 절개하여 간을 절제. 담낭을 제거하고 10cm 조직 배양 접시에 간장을 전송합니다. </li><li> 바이오 안전성 캐비닛에서 간으로 도금 중간 10 ㎖ (표 1)를 추가합니다. 조심스럽게 간세포를 제거하거나 집게 또는 메스를 사용하여 간을 긁어. 50 ㎖ 원뿔형 튜브에 100 ㎛ 세포 스트레이너 및 전달을 이용하여 현탁액을 필터. 50 ML 원뿔 튜브에 추가로 10 도금 중간 ㎖의 풀과 접시를 씻으십시오. </li><li> 350 XG, 4 ° C에서 5 분 동안 원심 분리하여 세포 펠렛. </li><li> 90 % 콜로이드 실리카 10ml를 PO 코팅 대기음 매체 도금 10 ml의 배지에 재현 탁 펠릿 및 추가lyvinylpyrrolidone. 단계 2.11에서와 같이 부드럽게 원심 분리기 섞는다. 생균이 아래쪽으로 펠렛 것이다 동안 원심 분리 후, 사균의 층은, 혼합물의 상부에 부유한다. </li><li> 기음 죽은 세포와 매체. 도금 중간, 2.11에서와 같이 원심 분리기 20 ㎖로 2 회 반복한다. </li><li> 도금 중간 10ml에 재현 탁 세포. 혈구와 세포를 계산하고 콜라겐 처리 된 24 웰 배양 접시에 9 × 104 세포 / 웰 놓습니다. 2 시간 동안 37 ° C 조직 문화 인큐베이터에서 품어. 각 플레이트는 지방산 산화 분석 OR 지방 생성 분석법 중 하나에 사용될 수있다. </li><li> 선택적으로, 37 ° C에서 유지 보수 매체 (표 1)과 문화에 우물을 변경합니다. 분석 결과에 영향을주지 않으면 서 3 일 - 2 세포를 배양 할 수있다. </li></ol> 3. 지방산 산화 분석 경고 : 방사능의 사용은 위험 할 수 있습니다. 모든 구매, 저장, 취급, 및 디방사성 물질의 폐기에이 기관, 주, 연방 정부의 규정 및 지침에 따라 수행되어야한다. <ol><li> 16-20 시간 이전에 분석에, 따뜻한 PBS로 세포를 2 회 반복한다. 20 나노 글루카곤과 혈청 혈청 기아 중간 (표 1)에 세포를 변경하고 하룻밤에 37 ° C를 품어. 참고 : 소 태아 혈청이 신진 대사 호르몬의 알 수없는 농도 (예를 들면 인슐린, 글루카곤)가 포함되어 있기 때문에, 혈청이 분석에 가질 수있는 혼란 효과를 제거하기 위해 세포를 굶어. 세포는&gt; 24 시간 동안 혈청 기아 배지에서 생존이다. 글루카곤 처리는 간세포에서 지방산 산화를 자극하는 데 사용된다. </li><li> 분석의 아침, 사전 인큐베이션 미디어를 준비 : 10 분 동안 70 ℃로 100 mM의 용액 및 열을 초순수 팔미틴산 나트륨 적당량 재현 탁. 한편, 필요한 양 (24- 웰 플레이트의 웰 당 0.5 ml)로 준비25 mM의 HEPES, 1 % BSA 분수 V, 20 nM의 글루카곤과 DMEM의. 37 ° C까지 가열한다. <ol><li> 가용화 후, 배지를 250 μM의 최종 농도 팔미 테이트를 추가한다. 중간 배양을 사전 2 시간 동안 37 ° C에서 부화하는 간세포를 변경합니다. 나중에 사용하기 위해 37 ° C에서 일부 사전 부화 매체를 보유합니다. </li></ol></li><li> 배양 중에, 질소 가스 하에서 증발에 의해 14 C-팔미틴산 적당량 (0.5 μCi를 / 웰)을 건조. <ol><li> 예를 들어, 24 웰 플레이트에 24 샘플을 측정하는 1.5 ㎖의 튜브에 0.1 μCi를 / ml의 14 C-팔미틴산 120 μL를 전송. 천천히 흄 후드 3-5 cm의 거리에서 솔루션을 통해 질소 가스를 불어 에탄올 용매를 증발. 상기 튜브의 하단에 건식 14 C-팔미두고 40 분 - 용매 약 30 증발한다. </li></ol></li><li> 약 15 분의 배양이 종료되기 전에, 14 재현 탁0.1 N 수산화 나트륨의 C-팔미 테이트 (12.5 μL / μCi를). 10 분 동안 70 ℃에서 배양한다. 따뜻한 사전 부화 중간의 세 권을 추가로 pipetting 아래로 섞는다. </li><li> 희석 (14) C-팔미 테이트의 25 μL 잘 각 스파이크. 부드럽게 판을 흔들어 혼합하고 90 분 동안 37 ° C에서 품어. 이것은 분석 플레이트이다. </li><li> 배양하는 동안, 필터 종이 접시 (그림 1)을 준비합니다. <ol><li> 멸균 24 웰 플레이트에서 커버를 제거합니다. 각 웰의 바닥에 종이 필터 중 하나 2cm X 2cm 조각을 놓습니다. 4 &quot;X 7&quot;파라 필름의 조각으로 접시를 오버레이. </li><li> 이러한 micropipettor 팁 박스로, 큰 직사각형의 물체를 사용하여, 웰 구멍에 관통하고 파라 필름을 플레이트의 나머지 위에 시일을 만들 웰에 파라 필름을 발라. 이제 우물을 덮고 파라 필름의 천공 원을 제거합니다. 플레이트는 지금 단단히 잘 O를 제외한 모든 지역에서 파라 필름으로 덮여해야한다penings. </li></ol></li><li> 10 분 배양이 끝나기 전에, 여과지 모든 액체를 흡수 확인한 여과지 플레이트의 각 웰을 3 N NaOH를 200 μL를 추가한다. <ol><li> 동결에 선택적으로 이전, 새로운 24 웰 플레이트에 매체를 전송합니다. PBS로 한번 세척 한 후, 0.1 N 염산의 나머지 세포 용균 및 BCA 분석으로 단백질 함량을 계산한다. </li></ol></li><li> 인큐베이션의 끝에, 액체 질소에서 플레이트 분석법 스냅 동결. 완전히 진행하기 전에 동결은 물론 각 보장하기 위해주의해야합니다. </li><li> 분석 플레이트의 각 웰에 70 % 과염소산 100 ㎕를 추가한다. 즉시 필터 종이 접시 커버. 2 시간 동안 실온에서 80 RPM의 궤도 속도로 궤도 흔드는과 바위에 판을 놓습니다. </li><li> 배양 후, 샘플 처리 : <ol><li> 섬광에 4 ㎖의 액체 섬광 유체 여과지 사각형을 전송, CO 2 분획을 측정유리 병 및 측정 (14) C 신호. </li><li> , 산 용해성 물질을 측정 1.5 mL의 미세 원심 분리 튜브에 매체의 400 μl를 전송합니다. 10 분 동안 최대 속도로 원심 분리기. 간단히 1 클로로포름 - 메탄올 (V / V)와 소용돌이 : 2의 500 μL에 상층 액 100 μl를 추가합니다. <ol><li> 다시 혼합물에 물을 250 μl를 추가하고, 소용돌이. 3,000 X g에서 10 분 동안 원심 분리기 샘플. 전사 섬광 바이알에 4 ㎖의 액체 섬광 유체 상에 상부 200 ㎕의 14 및 C 신호를 측정한다. </li></ol></li></ol></li></ol> 4. 지방 생성 분석 <ol><li> 저녁 전에 분석을 시작으로, 따뜻한 PBS로 세포를 2 회 반복한다. 100 nM의 인슐린과 혈청 기아 중간에 간세포를 변경합니다. 37 ° C에서 밤새 품어. 참고 : 소 태아 혈청이 신진 대사 호르몬의 알 수없는 농도 (예를 들면 인슐린가 포함되어 있기 때문에, 글루카곤을), 혈청이 검정에 가질 수있는 교란의 효과를 제거하기 위해 세포를 굶. 세포는&gt; 24 시간 동안 혈청 기아 배지에서 생존이다. 인슐린 지방 형성을 촉진하기 위해이 분석에서 사용된다. </li><li> 100 nM의 인슐린, 10 μm의 차가운 아세테이트 0.5 μCi를 3 H-아세테이트 당 잘 혈청 기아 매체 : 지방 생성 중간합니다. 지방 생성 중간에 세포를 변경하고 2 시간 동안 37 ° C에서 품어. 관심의 화합물을 테스트 할 포함합니다. </li><li> 잠복기 후, PBS로 세포를 2 회 반복한다. 0.1 N 염산 120 μL에 긁어를 Lyse 세포. 보호구 10 단백질 분석 용 μL (BCA 분석으로 평가) 및 전송 1.5ml의 미세 원심 분리 튜브에 100 μL. </li><li> (2) 500 μL를 첨가하여 추출 지질 : 1 클로로포름 - 메탄올 (v / v)로. 소용돌이는 간단히 5 분 동안 실온에서 품어. 물, 소용돌이의 250 μl를 추가하고 추가로 5 분 동안 실온에서 품어. 샘플을 원심 분리기 3000 XG, RT에서 10 분. 주의하여섬광 유리 병에 4 ml의 액체 섬광 유체에 낮은 위상을 전송하고 3 H 활성을 측정한다. </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.

관류에 희생에서 시간은 이상적인 관류 및 간 콜라게나 소화 미만 3 분이어야합니다. 관류 매체 관류가 시작되면, 간은 즉시 빨간색은 엷은에서에 모양을 변경해야합니다. LDM과 부화의 약 10 분 후, 간은 부어 핑크 나타납니다. 관류이 불충분하다는 경우, 간은 이러한 변화를 나타내지 않으며, 이는 일반적으로 낮은 수율 간세포 초래할 것이다. 세척 단계 후, 절연 간세포 전에...

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><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 &amp;deg;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&amp;nbsp;</td><td></td></tr><tr><td>Collagen</td><td>Life Technologies</td><td>A1048301&amp;nbsp;</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&amp;nbsp;</td><td></td></tr><tr><td>Tygon S3 Tubing&amp;nbsp;</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>24 G x 3/4&quot; 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&amp;rdquo; Straight, Blunt-Blunt Operating Scissors</td><td>Roboz</td><td>RS-6810</td><td>Pre-sterilize in autoclave</td></tr><tr><td>24 mm Blade Straight, Sharp-point Microdissecting Scissors</td><td>Roboz</td><td>RS-5912</td><td>Pre-sterilize in autoclave</td></tr><tr><td>4&amp;rdquo; 0.8 mm Tip Microdissecting Forceps</td><td>Roboz</td><td>RS-5130</td><td>Pre-sterilize in autoclave</td></tr><tr><td>4&amp;rdquo; 0.8 mm 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>CO&lt;sub&gt;2 &lt;/sub&gt;Incubator</td><td></td><td></td><td></td></tr><tr><td>Serological pipets</td><td></td><td></td><td></td></tr><tr><td>1,000, 200, 20 &amp;mu;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 총 세포 - 간세포 아이솔레이션는 일반적으로 1을 초래한다. 밤새 배양 한 후, 세포는 (그림 2) 이핵됩니다 많은 육각형를 나타납니다. 건강한 세포는 세포 죽음을 나타내는 있습니다 과립 또는 소포,없는해야한다. 일반적으로, 지방산 산화 분석은 시험 화합물 당 ​​3-4 회 반복 실행됩니다. CO 2 샘플 카운트 산 용해성 물질 유래의 약 1/5이...

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

드 노보 지방 생성 및 β - 지방산 산화는 간세포의 주요 대사 경로, 지방간 질환 등 여러 가지 대사 장애에 교란되는 경로를 구성한다. 여기에서 우리는 마우스 차 간세포의 분리를 보여와 β-지방산 산화 및 지방 생성의 정량화를 설명합니다.

마우스 차 간세포에서 지방산 산화 및 지방 생성의 결정

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 조회수.  University of Massachusetts Medical School.  드 노보 지방 생성 및 β - 지방산 산화는 간세포의 주요 대사 경로, 지방간 질환 등 여러 가지 대사 장애에 교란되는 경로를 구성한다. 여기에서 우리는 마우스 차 간세포의 분리를 보여와 β-지방산 산화 및 지방 생성의 정량화를 설명합니다. 

비디오: Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

갓 분리된 마우스 간세포의 현탁액에서의 지방산 β산화의 측정

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

연구

생화학

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

의 최적화 분석<em&gt; 생체</em&gt; 및<em&gt; 체외</em&gt; 간 지방증

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

의학

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

비 방사성 L-azidohomoalanine 라벨 방법에 의해 초기 단백질 합성 분석에 대 한 기본 마우스 Hepatocytes의 절연

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

분화된 HepaRG 세포에서 베아토시스 유도 및 특성화

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

마우스에서 전신 지질 처리 용량 평가

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

This protocol is intended to be a tool to study steatosis and the molecular, biochemical, cellular changes produced by the over exposure of hepatocytes to lipids in vitro.

시험관내실험용 스테토시스 모델 : 지질 과부하 조절 배지의 간세포 배양

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

마우스 원발성 간세포 분리를 위한 개선된 시간 및 노동력 효율적인 프로토콜

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

생물학

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large extracellular matrix proteins that compose bone tissue and then concentrate the ions necessary for its mineralization. Data on the metabolic requirements of bone formation are emerging rapidly. While much remains to be learned, it is expected that derangements in the intermediary metabolism contribute to skeletal disease. Here, a protocol is outlined to assess the capacity of osteoblastic cells to oxidize 14C-labeled fatty acids to 14CO2 and acid-soluble metabolites. Fatty acids represent a rich-energy reserve that can be taken up from the circulation after feeding or after their liberation from adipose tissue stores. The assay, performed in T-25 tissue culture flasks, is helpful for the study of gene gain or loss-of-function on fatty acid utilization and the effect of anabolic signals in the form of growth factors or morphogens necessary for the maintenance of bone mass. Details on the ability to adapt the protocol to assess the oxidation of glucose or amino acids like glutamine are also provided.

The present protocol describes an assay to assess the capacity for fatty acid oxidation in cultures of primary bone cells or relevant cell lines.

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large ...

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

생체 외 쥐 지방 조직 및 in vitro에서 분화된 1차 지방전구세포에서 지방분해 속도 측정

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

murine white 지방 조직의 반자동 분리

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

저자 스포트라이트: 조직 해리제를 사용하여 Murine White 지방 조직에서 기질 혈관 분획의 반자동 분리  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

조직 해리기를 사용하여 쥐 백색 지방 조직에서 기질 혈관 분획의 반자동 분리

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...