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 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 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

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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

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.

지방산의 정량 Phosphoproteomics은 자극 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 ...

연구

생물학

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.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

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.

토양에 미생물 잔류 결정을위한 Aldononitrile 아세트산 Derivati​​zed 글루코사민과 Muramic 산성의 GC 기반 탐지

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%.

쥐 간장 세포의 분리 및 차 문화

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;

기본 마우스 조골 세포에서 골 세포 배양을 구하는 레티노 산의 적용

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.

차 인간 간세포의 분리 및 비 실질 간 세포의 주요 집단 대응을위한 프로토콜

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.

격리 된 작업 쥐의 심장에서 포도당과 지방산 산화의 가격의 결정 방법

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.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

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.

체외에서 담즙 관내 형성에서 세포골격의 역할을 연구하기 위해 1 차 마우스 간세포의 격리 및 3D 콜라겐 샌드위치 문화

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.

다중 파라미터 관류 조절을 통한 원발성 쥐 간세포의 분리

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