우리는 토론, 기술 지원 및 원고에 대한 편집 지원 Haruya 오노, Kosaku 시노다, 루이스 샤프, 에미 Tomoda, 로렌 루이즈 감사드립니다. 이 작품은 돌파구 바이오 메디컬 연구 및 Asubio 알약 주식회사에서의 프로그램에서 SKULA에 NIH (DK087853)에서 보조금 코펜하겐과 EU FP7 프로젝트 DIABAT (건강-F2의 대학에서 SHARE 박사 장학금이 지원 한에 의해 지원되었다 리자 매드슨와 Karsten Kristiansen에 -2011-278373). 우리는 또한 DERC 센터 보조금 (NIH P30 DK063720)을 인정합니다.

1. 소화 매체를 준비 조직 당 5 마우스 (약 1 ML / 1g의 지방 조직) 당 5 ML하십시오. <ol><li> 소화 효소에 무게 : - Collaginase D : 1.5 U / ML (1108874103, 1 G, 로슈, 70334223) - Dispase II : 2.4 U / ML (04,942,078,001, 0980 MG / lyo, 로슈, 11466200) </li><li> 25 ML PBS를 추가하고 분해하기 위해 잘 섞는다 </li><li> 10 mm의 최종 농도에서 조직의 소화 직전에 CaCl 2를 추가 </li></ol> 2. 마우스에서 지방 조직을 해부 <ol><li> 절개는 신중하게 수행해야하고, 즉시 생쥐 후 (6~8주 세) 희생되어 있습니다. 을 열기 전에 마우스 모피에 뿌려 에탄올로 살균 작업 할 수 있습니다. 깨끗한 PBS, 별도의 interscapular BAT와 피하 사타구니 와트에 직접 조직을 놓으십시오. </li><li> interscapular BAT의 경우 : 최대 직면 뒷쪽으로 배치 마우스를 뒷면과 목의 모든 길을 따라 자르면. 갈색지방 조직이 바로 어깨 (interscapular) 사이의 피부에서 볼 수 있습니다. BAT는 두 엽, 모양의 나비로 볼 수 있습니다. BAT를 덮고 흰 지방의 얇은 층은 신중하게 제거 할 것입니다. </li><li> 피하 와트 (사타구니 와트)의 경우 : 피부를 제거합니다. 사타구니 와트는 내부의 고환을 향해 허벅지 아래로 거의 뒷면에 시작과 함께 가고, 양쪽의 피부 바로 아래에 자리 잡고 있습니다. </li></ol> 3. 지방 조직을 잘라 소화 <ol><li> 빠르게 머리, 골격 근육 및 조직 섹션에서 결합 조직처럼 모든 오염을 제거합니다. </li><li> PBS로 분해 매체를 희석하고 건조 판에 배치하지 않는 종이에 신속하게 조직을 말린다. </li><li> 소화 매체 (사전 소화에 CaCl 2 추가) 및 작은 조각으로 조직을 잘게 썬 고기를 추가합니다. 5 ML의 피펫과 등을 아래로 pipetting하여 잘 섞는다. 소화 m의 나머지 50 ML 튜브에 다진 조직을 전송edium은 아래로 pipetting과가 더 섞는다. </li><li> 다이제스트 37 ° C 40-50 분 150 rpm으로 일정하게 교반을 갖추고 있습니다. 소화가 잘되어 가고 이상 - 소화을 방지하기 위해 있는지 확인하기 위해 최선을 10-15 분을 확인합니다. </li></ol> 참고 : 올바른 소화 매체와 시간이 중요합니다. 조직이 잘 소화해야하지만, 초과 소화는 세포를 손상시킬 수 있습니다. 4. 필터 세포 현탁액 <ol><li> 5 ML 전체 매체 (DMEM/F12가 포함 된 10 %의 FPS와 P / S)를 추가하여 소화를 중지합니다. 세포는 거의 동질해야합니다. pipetting으로 잘 섞는다. </li><li> 10 분 700 XG에 원심 분리기. </li><li> SVF는 이제 튜브의 바닥에 갈색 펠렛으로 볼 수 있습니다. 상단과 액체 층의 대부분에있는 기름 성숙한 지방 세포 층을 대기음 - 펠렛을 유지하고 10 ML 완전한 중간에 용해. 잘 섞는다. </li><li> 새로운 50 셀 스트레이너 (50-70 μm의 직경을) 장소<html> ML 튜브와 세포 현탁액을 필터링합니다. </li></ol> 5. 판 셀 <ol><li> 10 분 700 XG에서 15 ML 튜브와 원심 분리기에 세포 현탁액을 전송합니다. </li><li> 10 ML 완전한 매체에 매체를 대기음하고 다시 일시 중지 펠릿. 피펫 잘 섞는다. </li><li> 필요한 얼마나 많은 접시 견적. 이 다양하고 펠렛의 크기에 따라 달라집니다 수 있습니다. 일반적인 규칙은 사타구니 와트 5 마우스와 BAT 한 10cm 판에서 10cm 플레이트 2입니다. </li><li> 완전한 중간에 콜라겐 코팅 요리에 플레이트 세포 (R &amp; D) </li><li> 1-2 시간 전지, 대기음 매체를 도금 후, 두 번 PBS로 씻어와 신선한 매체를 추가 할 수 있습니다. 이 적혈구, 면역 세포 및 기타 오염 물질을 제거 할 수 있습니다으로이 단계는 중요합니다. </li></ol> 6. 셀을 차별화 <ol><li> 유지 보수 및 유도 매체를합니다. </li></ol> 유지 보수 매체 추가 된 미디어를 작성하십시오 다닐 &quot;&gt; 인슐린, 최종 conc 5. μg / ML (5 MG / ML 주식, 산성화 물 산도에게 2.5를 준비하는 10 ML H 2 O의 아세트산의 *** 100 μl. 산성화 물에 인슐린을 분해. 스토어 재고에서 -20 ° C) 3,3 &#39;,5-Triiodo-L-thyronine (T3), 최종 conc. 1 나노 미터 (10 μM 재고, ***는 1N NaOH에 T3 분해 및 10 μM 주식을하기 위해 매체를 추가 할 수 있습니다. 시그마 고양이 # T-2877) 4 번 스토어 ° C, 일주일 동안 좋은 유도 매체 다음 화합물을 포함하는 유지 보수 매체 : 인도 메타 신, 최종 conc. 125 μM (에탄올 0.125 M 주식, 시그마 고양이 # I-7378). 인도 메타 신은 60로 가열해야 ° C 용해되어 있어야합니다. Dexamethasone, 최종 conc. 2 μg / ML (에탄올의 2 밀리그램 / ML 양말, 시그마 고양이 # D-1756) 3 - 이소 부틸-1-methylxanthine (IBMX), 최종 conc. 0.5 ㎜ (DMSO의 0.25 M 재고, 시그마 고양이 # I-5879) Rosiglitazone, 최종 conc. 0.5 μM(DMSO 10 MM 주식, 시그마 고양이 # R-2408) 참고 : 때마다 중간 신선한 유도하십시오. <ol start="2"><li> 전체 미디어에서 95~97%의 합류 (합류하지만 너무 포장되지 않음)에 기본 adipocytes 성장 </li><li> 유도 매체 (일 0)와 함께 정기적으로 전체 매체를 변경 </li><li> 48 시간 (2 일) 후, rosiglitazone (0.5 μM)와 유지 보수 매체로 매체를 변경 </li><li> 추가 48 시간 (4 일) 후, 추가 2-3 일 동안은 rosiglitazone과 신선한 유지 보수 매체 (1 μM)로 변경합니다. 6-7일 유도 매체를 추가 한 후, 세포가 완전히 지방 세포를 성숙하고 차별화되어 있으며 기름 방울로 가득합니다. 작은 물방울이 유도 한 후 3-4일 주위에 나타납니다. </li></ol> 참고 : 변경 매체는 세포가 완전히 구별 모든 2~3일 때까지. 전지는 지금 수확하고 Ucp1와 인접의 mRNA 표현 될 수있다R 갈색 지방 세포에 특정한 유전자가 qRT-PCR로 측정 할 수 있습니다. 서양 얼룩은 단백질을 감지하는 데 사용됩니다.

<ol>
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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+the+nuclear+receptor+peroxisome+proliferator-activated+receptor+gamma+promotes+brown+adipocyte+differentiation.">Activation of the nuclear receptor peroxisome proliferator-activated receptor gamma promotes brown adipocyte differentiation.</a> The Journal of Biological Chemistry. 271, 29909-29914 (1996).</li><li>Sugii, S., 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=PPARgamma+activation+in+adipocytes+is+sufficient+for+systemic+insulin+sensitization.">PPARgamma activation in adipocytes is sufficient for systemic insulin sensitization.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 22504-22509 (2009).</li></ol>

제작자들은 더 경쟁 금융 이익이 없다는 점 선언합니다.

여기 마우스의 기본 양식 adipocytes에 베이지 색 / brite 세포의 개발을 연구 할 수있는 신뢰할 수있는 셀룰러 시스템을 제시한다. 같은 몇 가지 가능한 불후의 세포 라인에 비해,이 시스템은 생체의 흰색 지방의 갈변으로 향상된 관련성을 제공 할 것으로 예상됩니다. 이러한 기본 adipocytes의 연구는 몇 가지 장점을 제공하더라도,이 또한 고려하는 것이 중요 일부 제한 사항...

<html> 비만은 크게 증가하고 세계이며, 지금은 공중 보건 1 가장 심각한 문제 중 하나를 간주됩니다. 이 조건은 지출에 대한 에너지 흡입 상대의 misbalance과 흰색 지방 조직 (와트)의 지질로 저장 초과하는 에너지의 결과에 관련되어 있습니다. 갈색 지방 조직은 열을 생산하기 위해 과도한 에너지를 발산 할 수있는 능력을 가지고있는 동안 확대 와트는 증가 몸 질량 및 무게와 연결되어 있습니다. 따라서 BAT은 춥고 비만 2,3 모두에 대한 보호 역할 할 수 있습니다. 이것은 단백질 1 (UCP1)를 uncoupling에 의해 미토콘드리아의 전자 교통 uncoupling에 의해 달성된다. 이 단백질은 BAT 3 thermogenesis를 nonshivering의 상징으로 간주됩니다. 최근 몇몇 연구는 성인 인간이 기능 BAT 4-8이 있으며, 따라서 인간의 BAT의 조작은 비만과 관련 질병과의 전투에서 잠재적 인 치료 개입 할 수 있다는 밝혔다. jove_content는 &quot;&gt; 현재 증거가 갈색 adipocytes의 두 종류가 동물에 존재임을 나타냅니다;&quot;고전 &quot;또는&quot;기존의 &#39;갈색 지방은 태아 단계에서 개발 및 interscapular 지역과 다른 주변 조직에 헌신 갈색 지방 창고를 형성 반면. , 갈색 지방 (그래서 brite 또는 베이지 색 세포라고도 함)의 &quot;inducible&quot;형태는 사후 산후 단계에서 개발과 흰색 지방 조직에 사이 사이에 나타납니다. 갈색 adipocytes의 두 종류도 다른 발달 기원으로 구분되어 있습니다. 기존의 동안 갈색 adipocytes가 Myf5 전구체 myoblastsic 같은으로부터 발생, 와트 사이 사이 inducible 베이지 / brite 전지는 비 Myf5 계보 (Lineage) 9,10에서 발생. 또한,이 세포 유형의 규제 경로는 Myf5 파생 갈색과 다를 수 가능성이 adipocytes 11. 베이지 색 세포 (흰색 지방의 예 &quot;브라우닝&quot;)의 개발은 만성 감기에 노출과에 대한 응답으로 활성화 할 수 있습니다β3-adrenoceptor-agonists 또는 성인 12-14에서 PPARγ의 agonists. 베이지 / brite 전지는 전체 에너지 균형의 조작을위한 유망 치료 표적이 될 가능성이 잠재적으로 비만 치료의 일부가 될 수 있습니다, 따라서, 환경 단서는 베이지 색의 개발을 제어하는​​하여 정확한 분자 메커니즘 및 신호 전달 경로를 이해하는 것이 중요합니다 세포. 흰색 지방의 갈변의 분자 제어를 이해하려면, 체외 실험에서 가장 preadipocytes 아니라 비동기 적으로 이루어집니다의 차별화로 적합하며 원위치 15 세포를 감지하기가 어렵습니다 수 있습니다. 지방 세포 개발에 연구가 지금까지 이러한 3T3-L1, 3T3-F442A 또는 HIB1와 같은 세포 라인에 주로 수행 된 있지만, 이러한 세포 라인은 베이지 색 세포의 분자 서명을 부족 나타납니다. 한편, 피하 와트로부터 격리 기본 adipocytes는 proces을 요점을 되풀이 할 가능성이 가장 높은셀 자치 패션의 흰색 지방의 갈변의 s입니다. 여기 지방 조직에서 stromal-혈관 분수의 효과적인 격리와 PPARγ의 agonists에 대한 응답으로 흰 지방의 갈변을 유도를위한 프로토콜을 제공합니다. Rosiglitazone은 이러한 세포의 갈변의 특히 효과적인 중재자로 표시되었습니다. 이전 16 제안으로 휴대폰이 시스템은 베이지 색 / brite 세포의 개발을 연구 할 수있는 신뢰할 수있는 셀룰러 시스템을 제공하는 데 사용할 수 있습니다.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 시약 </td></tr><tr><td> Collaginase D </td><td> 로슈 </td><td> 11088874103 </td><td></td></tr><tr><td> Dispase II </td><td> 로슈 </td><td> 04942078001 </td><td></td></tr><tr><td> CaCl 2 </td><td></td><td></td><td></td></tr><tr><td> DMEM 매체 </td><td> 어부 </td><td> 10017-CV </td><td> 나트륨 pyruvate없이 2,5 g / L 포도당 및 L-글루타민과 함께 </td></tr><tr><td> 인슐린 </td><td></td><td></td><td></td></tr><tr><td> T3 (3,3 &#39;,5-Triiodo-L-thyronine) </td><td> 시그마 </td><td> T-2877 </td><td></td></tr><tr><td> 인도 메타 신 </td><td> 시그마 </td><td> I-7378 </td><td></td></tr><tr><td> Dexamethasone </td><td> 시그마 </td><td> D-1756 </td><td></td></tr><tr><td> IBMX </td><td> 시그마 </td><td> I-5879 </td><td></td></tr><tr><td> Rosiglitazone </td><td> 시그마 </td><td> R-2408 </td><td></td></tr><tr><td></td><td></td><td></td><td> 장비 </td></tr><tr><td> 콜라겐 코팅 요리 </td><td> BD </td><td> 354450 </td><td> 10cm 플레이트 </td></tr><tr><td> 70 μm 필터 </td><td> BD 팔콘 </td><td> 352350 </td><td> 셀 스트레이너, 70 μm 나일론 1/ea </td></tr></tbody></table>

isolation and differentiation of stromal vascular cells to beige/brite cells

브라운 adipocytes는 미토콘드리아의 호흡 사슬을 풀다 및 열 등의 화학 에너지를 발산 할 수있는 능력을 갖추고 있습니다. 흰 지방 조직 (그래서 베이지 색 또는 brite 세포라고도 함)의 UCP1 - 긍정적 인 갈색 adipocytes의 개발은 매우 같은 만성 감기에 노출 또는 PPARγ의 agonists의 환경 단서의 다양한 의해 유도되며, 따라서,이 세포 유형에 대한 치료 대상으로 잠재력이있다 비만 치료. 대부분의 불후의 지방 세포 라인 문화에 흰색 지방의 &quot;브라우닝&quot;의 과정을 요점을 되풀이 할 수는 없지만, 기본 adipocytes는 brite / 베이지 색 세포 개발의 분자 제어를 연구하기위한 신뢰할 수있는 셀룰러 시스템을 제공 피하 화이트 지방 조직에서 stromal 혈관 분율 (와트)에서 분리 . 여기 주요 preadipocytes을 효과적으로 격리와 문화에 베이지 / brite 셀에 차별화를 유도를위한 프로토콜을 설명합니다. 브라우닝 효과는 갈색 지방 선택 마르크의 표현에 의해 평가 될 수있다같은 UCP1과 같은 ERS.

주요 adipocytes의 브라우닝은 mRNA의 Ucp1의 표현과 qRT-PCR로 다른 갈색 지방의 특정 또는 선택적으로 유전자를 측정하여 액세스 할 수 있습니다. 그림 1에 제시하는 것은 사타구니 와트 - 파생 주요 adipocytes에서 유전자 발현 데이터입니다. 세포는 각각 50 nm의 500 nm의에서 rosiglitazone 두 가지 복용의 면전에서 차별화 유도했다. 셀의 하위 집합을 수확하기 전에 4 시간 10 μM에서 forskolin?...

Watch this Scientific Journal Video about Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells at JoVE.com

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

마우스의 흰색 지방 조직으로부터 격리 기본 흰색 preadipocytes는 베이지 색 / brite 세포로 구분 할 수 있습니다. 여기에 제시된 것은 백색 지방의 &quot;브라우닝&quot;의 분자 규정을 연구 할 수있는 신뢰할 수있는 셀룰러 모델 시스템입니다.

베이지 / Brite 셀에 Stromal 혈관 세포의 분리와 차별화

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

isolation-differentiation-stromal-vascular-cells-to-beigebrite

Research

JoVE Journal

Biology

38.4K 조회수.  University of California, San Francisco. 마우스의 흰색 지방 조직으로부터 격리 기본 흰색 preadipocytes는 베이지 색 / brite 세포로 구분 할 수 있습니다. 여기에 제시된 것은 백색 지방의 "브라우닝"의 분자 규정을 연구 할 수있는 신뢰할 수있는 셀룰러 모델 시스템입니다.

비디오: Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

인간 탯줄의 분리 및 동물 혈청 무료 확장 Mesenchymal Stromal 세포 (MSCS)와 내피 콜로니 형성 전구 세포 (ECFCs)을 유래

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

연구

생물학

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

간엽 전환을 유도하고 상피의 분석

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

분리 및 마우스 대동맥 내피 세포의 차 문화

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146의 분리<sup&gt; +</sup쥐 폐에서&gt; 주민 폐 중간 엽 기질 세포

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

쥐 관상 동맥 혈관 평활근 세포의 분리

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

동적 세포 돌출부에서 소프트웨어를 이용한 단백질 농도 추적을위한 그래픽 사용자 인터페이스

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

절연, 특성, 및 성인 마우스 심장에서 심장 줄기 세포의 분화

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

붉은털원숭이 지방유래 줄기세포의 분리, 증식 및 분화

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ 과도 현상 및 L형 칼슘 전류의 동시 측정을 위한 고품질 쥐 심방 및 심실 근세포의 분리

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...