이 작품은 NIH (R01HL128714 CF에) 및 (KS에 F31DK10236101)와 미국 심장 협회 (CF와 AG)에 의해 지원되었다.

모든 동물 프로토콜과 실험 절차는 의학의 마이애미 밀러 학교의 대학 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 1. 준비 <ol><li> 데우고 간 관류 미디어 간 37 ℃에서 수욕 미디어 다이제스트. </li><li> 관류 펌프 (30 mL / 분)를 설정합니다. 조심스럽게 간 관류 미디어와 펌프의 배관 시스템을 미리 기입. 시스템의 모든 공기 방울을 피하고 정위 현미경을 준비합니다. </li><li> I 제조사의 프로토콜에 따라 콜라겐 타입을 사용 외투 세포 배양 접시. </li></ol> 2. 간 복구 <ol><li> 산소 / 흡입 이소 플루 란 (이소 플루 4 % / 2 L / min의 산소)를 이용하여 공여 마우스를 마취. <ol><li> 2-2.5 %의 이소 플루 란을 저하 또는 마취제를 주입하여 마취를 유지한다 (100 ㎎ / ㎏ 복강 신체 체중)과 자일 라진 (체중이 10 ㎎ / kg intraperitoneally). 연속 이소 플루 란 마취 케타민 혈압을 낮추고 간독성을 유발할 수 있기 때문에 바람직하다. </li></ol></li><li> 흡수 패드에 마취 마우스를 놓습니다. 접착 테이프로 마우스의 사지를 고정합니다. </li><li> 면도와 마우스의 복부를 소독하고 선명한 / 무딘 팁 수술 가위를 사용하여 간의 두개골 경계에 치골에서 복부 개복술을 수행합니다. 양측 복벽, 진동판의 꼬리를 절개. </li><li> 심하게 우측으로 부위를 이동하여 하대 정맥 (IVC)을 노출. 조심스럽게 멸균 솜 팁 면봉와 각도 팁 집게를 사용하여 IVC 주변의 지방 조직을 해부하다. </li><li> suprahepatic 다이어프램을 절개하고 선명한 / 날카로운 팁 미세 수술 가위를 사용하여 두 개의 횡 절개하여 흉강을 엽니 다. 흉부 IVC는 수술 실크 (5/0)를 사용하여 결찰. </li><li> 조심스럽게 바늘을 제거, 차폐 정맥 카테터를 사용하여 infrarenal IVC를 Cannulate관류 펌프를 연결하고 간을 관류 시작합니다. 제대로 수행 한 경우, 간은 즉시 희게하고 팽창하기 시작한다. </li><li> 미세 수술 가위를 사용하여 혈액 및 관류 미디어의 적절한 배수를 허용 정 맥 포털을 가로로 쪼개다. </li><li> 간 관류 매체의 약 30 ㎖로 간을 관류하고 용지 (30 ㎖)를 소화 간 전환. 관류 펌프를 끄고 관류가 완료되면 정맥을 제거합니다. </li><li> 부드럽게 간의 중심 영역을 노출하고 간 로브를 연결하는 연결 조직을 찾습니다. 중앙 연결 섬유를 잡고 조심스럽게 자리에 간을 들고 모든 조직을 해부 살짝 간을 제거합니다. </li><li> 즉시 소화 매체 간 15ml를 함유하는 50 ㎖ 폴리 프로필렌 원추형 튜브 간을 옮긴다. 참고 :이 단계에 대한 구체적인 타임 라인이 없습니다. 재관류 후, 간은 무균 세포 배양 후드에 전송을 위해 다이제스트 미디어에 남아 있습니다. 모든이야ubsequent 단계가 무균 환경에서 수행되어야한다. </li></ol> 세포의 3. 분리 및 치료 <ol><li> 세포 배양 후드 내 멸균 조직 배양 접시에 50 ㎖ 폴리 프로필렌 원추형 튜브 함유 소화 매체 간을 옮긴다. </li><li> 멸균 포셉 사용하여 부드럽게 간의 네 로브 눈물. 제대로 수행 한 경우, 소화 매체 인해 간에서 간세포를 분리에 혼탁이 될 것이다. </li><li> 25 mL의 멸균 피펫 팁을 사용하여, 소화 조직 입자 및 세포 파편을 제거하기 위해 새로운 50 ㎖ 폴리 프로필렌 원추형 튜브에 70 ㎛의 나일론 세포 여과기를 통해 혼탁 소화 매체 필터. </li><li> 반복 3.2 멸균 인산 완충 식염수 (PBS) 3.3 단계. 이는 간에서 잔류 간세포를 분리하여 지원한다. </li><li> 4 ℃에서 2 분 50 XG에 원심 분리기. 25 mL의 차가운 1X 세포를 다시 일시 중지 부드럽게 초과 세포 파편을 포함 뜨는을 기음과윌리엄스 &#39;중간 E. </li><li> 25 mL의 멸균 피펫 팁을 사용하여, 청소기 현탁액을 달성하기 위해 50 ㎖ 폴리 프로필렌 원추형 튜브에 새로운 70 ㎛의 나일론 세포 여과기를 통하여 재 현탁액을 필터. </li><li> 반복 3.5 및 명확한 세포 현탁액을 얻기 위해 추운 윌리엄스 &#39;중간 E 1X를 사용하여 세 개의 추가 세척 3.6 단계를 반복합니다. </li><li> 최종 세척 단계 후, 흡 상등액 초과 균체 잔사를 제거했다. 다시 일시 중지하고 50 mL의 폴리 프로필렌 원뿔 관에 새로운 70 μm의 나일론 셀 스트레이너를 통해 차 간세포 해동 및 도금 보충제를 포함 25 mL의 따뜻한 윌리엄스 &#39;미디어 E 1 배속에서 필터 세포. </li><li> 혈구를 사용하여 25 ㎖의 세포 현탁액 내의 셀 개수. 성인 간 당 2000 만 셀 - 평균적으로 15을 기대합니다. 트리 판 블루 염색을 사용하여 세포 생존을 결정합니다. </li><li> 콜라겐 코팅 6 잘 가전에 차 간세포 해동 및 도금 보충제를 포함 2 mL의 &#39;윌리엄스의 미디어 E 1 배속에서 플레이트 세포LL 문화 클러스터 (물론 당 9 × 10 5 세포). </li><li> 4 시간 후, 차 간세포 유지 보수 보충제를 포함 윌리엄스 &#39;미디어 E 1 배속 미디어를 변경합니다. </li><li> 24 시간 후, 혈청 둘 베코의 변형 이글 배지 (DMEM) 미디어 1 배 6 시간 동안 세포를 굶어. </li><li> 차량 FGF23 (25 NG / ㎖), LPS로 PBS로 세포를 처리 (100 μg의 / ㎖) 또는 무 혈청 배지에서 IL-6 (50 ng의 / ㎖) 및 24 시간 동안 배양한다. </li></ol> RNA 4. 분리 <ol><li> 제조업체의 지시에 따라 상용 스핀 컬럼 키트를 사용하여 RNA를 준비한다. </li></ol> 역전사에 의해 고립 된 RNA 5. 생성 된 cDNA <ol><li> PCR의 튜브에 고립 된 RNA 샘플의 200 NG를 추가합니다. </li><li> 단계 5.1에서 효소가없는 H 2 O PCR에 튜브 14 μL를 추가합니다. </li><li> 단계 5.1에서 PCR 튜브에 역전사 효소의 4 μL를 추가합니다. </li><li> 혼합물이 균일하게 부드럽게 내용을 다시 중단; 부드럽게 원심 분리기. </리&gt; <li> 열 순환기에 혼합물을 포함하는 PCR 튜브를 놓습니다. 실행 역전사에 대한 열 순환기 : 다음 25 ° C (5 분), 42 ° C (30 분)의 1주기, 85 ° C (5 분)와 4 ° C에서 개최합니다. 최종 제품은 원하는 cDNA를 할 것이다. </li></ol> 6. 정량적 실시간 PCR에 의한 세포의 분석 (qPCR에) <ol><li> 96 웰 플레이트를 준비합니다. <ol><li> 1 μL의 cDNA, 0.25 μM 정방향 프라이머, 0.25 μM 역방향 프라이머, 5 μL SYBR 녹색, 10 μL의 총 부피 3.5 μL PCR 등급 물 : 얼음에 qPCR에 반응의 모든 구성 요소를 배치합니다. SYBR 녹색을 사용하여, 세중의 모든 샘플을 실행하는 마스터 믹스를 준비합니다. </li><li> 마스터 믹스가 완료되면 소용돌이는 균질 확인합니다. 96 WLL 플레이트의 각 웰에 나누어지는 9 μL qPCR에의 마스터 믹스. 후 신중하게 잘 각각에 cDNA를 1 μL를 추가합니다. 총 반응 부피는 10 μL 될 것입니다. </li><li> 로딩이 완료된 후 광 96 웰 플레이트를 밀봉접착 필름 간단히 원심 분리기 96 웰 플레이트 (1 분 50 XG). </li></ol></li><li> 리얼 타임 PCR 기기에서 실행 샘플. 실시간-PCR 기기에 밀봉 된 96 웰 플레이트 및 장비 제조업체 권장 사항에 따라 실행 샘플을 놓습니다. 표준 및 빠른 사이클의 예는 아래에 포함되어 있습니다. <ol><li> 60 초 동안, 15 초 동안 다음, 120 초 동안 94 ° C에서 60 ° C를 94 ° C의 40주기를 초기 변성 한 다음 선택적으로 4 ° C에서 개최 : 표준 사이클 파라미터를 들어, 다음을 사용합니다. </li><li> 빠른 순환 매개 변수의 경우 : 5 초, 15 초 동안 58 ° C, 10 초 동안 다음 72 ° C에 대해 다음 30 초 동안 95 ° C의 40주기를 94 ° C에서 초기 변성 다음을 사용합니다. </li></ol></li><li> 데이터를 분석하는 방법에 대한 지침은 장비 설명서를 참조하십시오. </li></ol> ELISA에 의한 세포 상층 액 7. 분석 참고 : 제조사의 프로토콜에 따라 모든 단계를 수행합니다. </P&gt; <ol><li> 샘플 준비 <ol><li> 별도의 튜브에 희석제 398 μL를 분배. 398 μL 희석액 튜브에 세포 상층 액 샘플의 피펫 2 μL. 이것은 200의 희석 배를 제공 할 것입니다. </li><li> 각 샘플 단계를 반복 7.1.1 테스트 할 수 있습니다. </li></ol></li><li> 순서 <ol><li> 피펫 100 내지 200 배 희석 한 시료와 μL를 96 웰 플레이트의 웰에 표준. </li><li> 150 rpm에서 마이크로 플레이트 쉐이커상에서 45 분 동안 25 ° C에서 96 웰 플레이트를 인큐베이션. </li><li> 우물을 1 배 세척 완충액으로 5 회 반복한다. 마지막으로 세탁이 완료되면, 흡수지 모든 잔여 세척액을 제거한다. </li><li> 각 웰에 HRP-복합 시약 500 μL를 추가합니다. </li><li> 7.2.3 - 반복 7.2.2 단계를 반복합니다. </li><li> 테트라 메틸 벤지딘의 피펫 100 μL (TMB) 96 웰 플레이트의 각 웰에 시약. </li><li> 부드럽게 마이크로 플레이트에서 20 분 동안 25 ° C에서 96 웰 플레이트 믹스150 rpm에서 진탕. 직접 각 웰에 정지 용액 100 μL를 첨가하여 반응을 정지하고 부드럽게 혼합한다. 정지 용액을 첨가 한 후에 색깔이 황색 회전한다. 이것은 정지 용액 정확하게 혼합되었음을 알린다. </li><li> 미세 적정 플레이트 판독기를 사용하여, 처음 5 분 이내에 450 nm에서의 광학 밀도를 결정한다. </li></ol></li><li> 결과의 계산 <ol><li> 각각의 샘플 및 각 표준에 대한 평균 흡광도 값 (A 450)의 표를 계산합니다. </li><li> NG에 표준 농도에 대한 각 기준의 450 평균 음모를 꾸미고하여 표준 곡선을 조립 / 선형 그래프 mL로. X 축이 농도 및 450의 값을 나타내는 Y 축를 대표 할 수있다. </li><li> (가) 각 희석 된 시료에서 450 의미 이용하여, 표준 곡선으로부터 NG / ㎖에서 목적 단백질의 농도를 결정한다. </li><li> 사실상 희석하여 샘플의 농도를 곱아르 자형. 이것은 세포 상등액 시료 중에 목적 단백질의 실제 농도를 결정하기 위해 허용 할 것이다. </li><li> 선 그래프로 샘플에서 구성된 테이블을 플롯. OD 450 값이 표준 곡선의 밖에있는 경우, 샘플을 적절하게 희석해야하고 다시 테스트. </li><li> 500,000 셀에 결과를 정상화. </li></ol></li></ol>

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G., Rusyn, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+for+liver+toxicity+testing.">In vitro models for liver toxicity testing.</a> Toxicol. Res. 2 (1), 23-39 (2013).</li><li>Lee, S. M. L., Schelcher, C., Demmel, M., Hauner, M., Thasler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Human+Hepatocytes+by+a+Two-step+Collagenase+Perfusion+Procedure.">Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure.</a> J Vis Exp. (79), (2013).</li><li>Kegel, V., 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=Protocol+for+Isolation+of+Primary+Human+Hepatocytes+and+Corresponding+Major+Populations+of+Non-parenchymal+Liver+Cells.">Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells.</a> J Vis Exp. (109), (2016).</li><li>Moshage, H., 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=Cytokines+and+the+hepatic+acute+phase+response.">Cytokines and the hepatic acute phase response.</a> J Pathol. 181 (3), 257-266 (1997).</li><li>Klaunig, J. 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=Mouse+liver+cell+culture.+I.+Hepatocyte+isolation.">Mouse liver cell culture. I. Hepatocyte isolation.</a> In vitro. 17 (10), 913-925 (1981).</li><li>Lu, Y. C., Yeh, W. C., Ohashi, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS/TLR4+signal+transduction+pathway.">LPS/TLR4 signal transduction pathway.</a> Cytokine. 42 (2), 145-151 (2008).</li><li>Schmidt-Arras, D., Rose-John, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-6+pathway+in+the+liver:+From+physiopathology+to+therapy.">IL-6 pathway in the liver: From physiopathology to therapy.</a> J Hepatol. 64 (6), 1403-1415 (2016).</li><li>Grabner, 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=Activation+of+Cardiac+Fibroblast+Growth+Factor+Receptor+4+Causes+Left+Ventricular+Hypertrophy.">Activation of Cardiac Fibroblast Growth Factor Receptor 4 Causes Left Ventricular Hypertrophy.</a> Cell Metab. 22 (6), 1020-1032 (2015).</li><li>Shulman, M., Nahmias, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17,+Long-Term+Culture+and+Coculture+of+Primary+Rat+and+Human+Hepatocytes.">Chapter 17, Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes.</a> Methods Mol Biol. 945, 287-302 (2012).</li></ol>

저자는 선언하는 이익의 충돌이 없습니다.

마우스에서 차 간세포를 분리하면 염증 반응 생체를 연구하는 빠르고, 저렴하고 신뢰할 수있는 도구입니다. 정확하게 수행하면 결과가 쉽게 발생하고, 신속하고 비용 효율적인 방식으로 재생 될 수있다. 다음 사항을 신중 성공적인 차단을 보장하기 위해 평가되어야한다. 수술 절개와 IVC의 삽관은 전신 마취가 아닌 안락사 후 수행해야합니다. 젊은, 경험이 연구...

만성 신장 질환 (CKD)는 급성 및 만성 염증 (1)의 상태로 정의 될 수있다. CKD 환자에서 phosphaturic 호르몬 섬유 아세포 성장 인자 23 (FGF23)의 혈중 농도가 점진적 혈청 인산염이 항상성을 유지하기 위해 증가. 증가 된 혈청 FGF23 수준은 독립적으로 혈액 투석 치료 3, 4를 시작하는 환자 중 심혈 관계 이환율과 사망률과 연관되어 있습니다. 또한, 여러 임상 연구 상승 FGF23 수준 및 C 반응성 단백질 (CRP), 인터루킨 6 (IL-6) 및 종양 괴사 인자 α (TNFα) (5), (6)의 혈청 레벨 사이의 강한 상관 관계를 보여 주었다. 또한, 실험 연구에서, 우리는 최근에 FGF23 직접 간세포를 대상으로하고 증가 CRP와 IL-6 페이지에 의한 염증 반응을 일으킬 수 있다는 것을 증명하고있다간 7 roduction. 따라서, FGF23는 CKD에 전신 염증에 기여하는 순환 요인으로 작용할 수 있습니다. 70 년대 초, 차 간세포는 고립되었다 처음으로 8 공부했다. 이후 일차 배양 된 간 세포는 광범위하게, 열 처리는 대사 호르몬 기능 약물 대사 및 독성뿐만 아니라 면역 및 염증 반응을 조사 구하는데 사용되어왔다. 이전 프로토콜은 주로 인간 간 조직 (11) (12)로부터 일차 간세포의 효소 격리 설명 하였다. 우수한 모델이지만,이 복합 간 시그널링 메커니즘뿐만 아니라 자극의 종류에 따라 기능적 결과에 미치는 영향을 연구하는 유전자 조작 능력을 남긴다. 이하에서, 우리는 기본 쥐 간세포의 단리를 설명. 특히, 예컨대 리포 폴리 사카 라이드 (LPS), IL-6와 같은 FGF23 간 급성 단계 반응의 여러 매개체의 효과는 쉽게 빠르고 재현성있는 방법 (13)에 분석 될 수있다. 여기서, 우리는 성체 생쥐에서 간세포의 효소 격리를위한 프로토콜을 제공하고, 우리는 LPS 및 IL-6과 같은 FGF23 같은 신규 염증성 매개체로서 염증 확립 유도제는 직접 발현 및 분비를 자극 할 수 있음을 입증 이러한 배양 간세포 CRP 및 IL-6과 같은 염증성 사이토킨.

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induction of an inflammatory response in primary hepatocyte cultures from mice

간은 전신적인 염증 반응의 조절에 결정적인 역할을한다. 특히 만성 신장 질환, 간은 요독, 산화 스트레스, 내 독소 혈증 및 급성기 반응물의 대량 생산으로 염증성 사이토 카인 순환 감소 간격에 응답하여 반응한다. 염증 및 간세포의 기본 역할 실험 도구 연구는 만성 신장 질환으로 특히 복잡한 설정에서 규제와 간 사이토 카인의 기여 전신 급성 위상 응답 및 연장 염증성 시나리오를 이해하기 중요하다. 생체 내에서 염증의 복잡한 메커니즘을 연구하는 것은 도전 남아 있기 때문에, 리소스를 많이 사용하는 보통 및 형질 전환 동물의 사용을 필요로 차 격리 된 간세포는 간 급성 위상 응답으로 기계적인 통찰력을 얻을 수있는 강력한 도구를 제공합니다. 이 체외 기술은 적당한 비용을 갖추고 있기 때문에, 높은 재생산성 및 일반적인 기술 지식은 주 절연 간세포 쉽게 스크리닝 방법으로 사용될 수있다. 여기, 우리는 기본 쥐의 간세포를 분리하는 효소 기반의 방법을 설명하고, 우리는 ELISA 및 정량 실시간 PCR을 사용하여 이러한 세포에서 염증 반응의 평가에 대해 설명합니다.

조직학 기본 격리 및 배양 된 세포의 대표적인 광학 현미경 이미지는도 1a에 도시되어있다. 면역 조직 화학적 분석은 절연 간세포 높은 알부민 (적색)뿐만 아니라, 섬유 아세포 성장 인자 수용체 4 (FGFR4) (녹색)을 표현할 수 있음을 보여준다. 핵 4 &#39;, 6-diamidino-2-페닐 인돌 (DAPI) (파란색)로 염색된다. (그림 1B).</s...

Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

여기서는 성인 마우스로부터 일차 간세포를 분리하는 효소 방법을 보여, 우리는 ELISA 및 실시간 PCR을 이용하여 염증 반응을 정량 설명한다.

마우스에서 차 간세포 문화에 염증 반응의 유도

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

induction-an-inflammatory-response-primary-hepatocyte-cultures-from

Research

JoVE Journal

Immunology and Infection

9.9K 조회수.  University of Miami Leonard M. Miller School of Medicine.  여기서는 성인 마우스로부터 일차 간세포를 분리하는 효소 방법을 보여, 우리는 ELISA 및 실시간 PCR을 이용하여 염증 반응을 정량 설명한다. 

비디오: Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL 마우스의 실험적 면역 Hypophysitis의 유도

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

연구

면역학 및 감염병학

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

성인 간세포의 혼합 차 문화에서 Macrophages을 분리하는 간단하고 효율적인 방법

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

기본 인적 Keratinocytes에서 Organotypic 뗏목 문화의 생성

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

신생아 쥐 뇌 조직의 혼합 Glial 세포 배양의 기본 Microglia 격리

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

골수 이식으로 마우스 대장염에서 면역 및 비 면역 세포에서 유전자 발현의 기능 역할을 차별화

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Diabetogenic CD4의 입양 전송하여 마우스의 가속 제 1 형 당뇨병 유도 + T 세포

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

이펙터 CD4의 입양 전송하여 쥐과 장내 염증의 유도<sup&gt; +</sup&gt; CD45RB<sup&gt; 높은</sup&gt; 면역 결핍 마우스에게 T 세포

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

심사 분석 실험 소설 내 피 레 귤 레이 터 염증 반응에 관여 하

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

마비 및 비주얼 시스템 부상 Neuromyelitis Optica Autoantigen Aquaporin-4에 대 한 T 세포 관련 하 여 쥐에서의 유도

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...