我们感谢大野治哉，筱田耕作，路易·夏普，友田惠美，劳伦·鲁伊斯的手稿上的讨论，技术帮助和编辑协助。这项工作是支持的股份从哥本哈根与欧盟FP7：项目DIABAT（HEALTH-F2大学的博士研究生奖学金补助金从美国国立卫生研究院（DK087853），从程序突破性的生物医学研究和从Asubio医药公司SKULA的支持-2011-278373）莉莎·马德森和Karsten克里斯蒂安森。我们也承认DERC的中心授予（NIH P30 DK063720）。

1。准备消化液 5毫升，每5只小鼠组织（大约1毫升/ 1克脂肪组织）。 <ol><li>在消化酶称： - Collaginase D：1.5单位/毫升（1108874103，1克，罗氏，70334223） - 中性蛋白酶II：2.4单位/毫升（0980毫克/ LYO，罗氏，04942078001，11466200） </li><li>加入25毫升PBS，拌匀溶解</li><li>的最终浓度为10mM，加入CaCl 2的只是事先消化组织</li></ol> 2。解剖小鼠脂肪组织<ol><li>应仔细进行解剖，并立即牺牲后的小鼠（6-8周龄）。在打开它之前，用乙醇消毒喷洒鼠标上的毛皮工作。将组织直接在干净的PBS，独立的肩胛间BAT和皮下腹股沟WAT。 </li><li>肩胛间BAT：有鼠标放在面临的后面，沿后面，所有的方式向颈部切开。棕色可以找到的肩膀（肩胛间）之间的皮肤下脂肪组织。 BAT可以被看作是两叶，蝴蝶形。覆盖一层薄薄的白色脂肪的BAT应仔细清除。 </li><li>对于皮下，WAT（腹股沟WAT）：去除皮肤。腹股沟WAT直属双方在皮肤上，开始几乎就在后面，旁边，里面的大腿和向下的睾丸。 </li></ol> 3。剪切和消化脂肪组织<ol><li>快速删除所有的污染，如头发，骨骼肌肉和结缔组织的组织切片。 </li><li>干组织迅速在纸上中与PBS稀释解剖，并把它放在一个干板。 </li><li>加入消化液（添加氯化钙消化前）和切成小块，剁碎的组织。 5毫升的移液管移液和组合非常好。将切碎的组织在50毫升管传输的其余部分的消化米edium，混合均匀，上下吹打。 </li><li>在37°C的精华在150 rpm的转速下为40-50分钟，不断搅拌。检查每10至15分钟，以确保消化进展顺利，以防止过度消化。 </li></ol> 注意：正确的消化液和时间是很重要的。的组织需要很好地消化，但多余的消化可以对细胞造成损害。 4。过滤器细胞悬浮<ol><li>通过加入5毫升完全培养基（DMEM/F12中含有10％的FPS和P / S）终止消化。几乎完全同质的细胞。移液拌匀。 </li><li> 700×g离心10分钟，离心。 </li><li> SVF现在可以被看作是一种褐色的颗粒上的试管底部。吸取油状的成熟的脂肪细胞层的顶部和大部分的液体层 - 保留沉淀，并溶解在10毫升完全培养基。拌匀。 </li><li>一个新的50年，将一个单元格的过滤器（直径为50-70微米）<html>毫升管和过滤细胞悬浮液。 </li></ol> 5。板细胞<ol><li>细胞悬浮液转移到一个15毫升的试管，在700×g离心10分钟离心。 </li><li>吸液的介质中，并重新悬浮在10ml完全培养基沉淀。移液器拌匀。 </li><li>估计需要多少个盘子。这可能会有所不同，取决于颗粒的大小。一般的规则是从5的小鼠腹股沟疝WAT和一个10厘米的板BAT 10厘米的板。 </li><li>在完全培养基板细胞上胶原涂层的菜肴（R＆D） </li><li> 1-2小时电镀后的细胞，吸出培养基，用PBS洗两次，加入新鲜培养基。此步骤是很重要的，因为这可以去除红血细胞，免疫细胞和其他污染物。 </li></ol> 6。区分细胞<ol><li>维护和归纳介质。 </li></ol>维护介质完成培养基中加入： 耳鼻喉科“&gt;胰岛素，终浓度5微克/毫升（5毫克/毫升库存中，*** 100微升乙酸在10ml H 2 O以制备酸化的水的pH值2.5。溶解胰岛素的酸化水。商店股票-20°C） 3,3&#39;-2,5 - 三碘-L-甲状腺原氨酸（T3），最终浓度。 1纳米（10μM股票，***溶解T3 1N氢氧化钠和添加中，使10μM股票。西格玛猫＃T-2877） 储存于4℃下，良好的一个星期诱导培养基维持培养基含有下列化合物： 吲哚美辛，最终浓度。 125微米（0.125 M的股票乙醇，适马猫＃I-7378）。吲哚美辛，必须加热至60℃进行溶解。 地塞米松，最终浓2微克/毫升（2毫克/毫升的袜子在乙醇中，Sigma公司猫＃D-1756） 3 - 异丁基-1 - 甲基黄嘌呤（IBMX），最终浓度。 0.5毫米（0.25 M DMSO，Sigma公司股票在猫＃I-5879） 罗格列酮，终浓度。 0.5μM（10 mM的股票在DMSO，Sigma目录＃R-2408） 注意：每次作出新的诱导培养基。 <ol start="2"><li>原代脂肪细胞生长至95-97％汇合，在完全培养基（汇合但不能太包装） </li><li>变化规律完全培养基诱导培养基（第0天） </li><li>经过48个小时（2天），变更中维持培养基中，罗格列酮（0.5μM） </li><li>额外的48小时（4天）后，改变与罗格列酮（1μM）2-3天的新鲜维持液。加入诱导培养基后6-7天，细胞完全分化成熟的脂肪细胞，并填充有油滴。诱导后，液滴周围会出现3-4天。 </li></ol> 注意：更改介质每隔2-3天，直至完全分化的细胞。 现在可以收获细胞mRNA的表达UCP1和其他可通过定量RT-PCR·布朗脂肪细胞特异性基因。 Western blot分析将被用于检测蛋白质。

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作者宣称，他们没有竞争的金融利益。

在这里，我们提出了一个可靠的蜂窝系统研究的发展，米色/ BRITE细胞的原代培养的脂肪细胞在小鼠体内。几个可用的永生化细胞系相比，这个系统是有可能提供增强相关的的白色脂肪在体内的褐变。 即使这些原代脂肪细胞的研究提供了一定的优势，也存在着一定的局限性和关切问题，是重要的考虑因素。首先，这个系统是高度依赖的细胞的分化能力。如UCP1和<em...

<html>肥胖是显着世界范围内不断增加，现在被认为对公众健康1的最严重的问题之一。这种情况涉及到的能量摄入相对misbalance支出和结果，多余的能量储存在白色脂肪组织（WAT）的脂质。扩大WAT与增加身体的质量和重量，而棕色脂肪组织的能力，以消耗多余的能量以产生热量。因此，BAT可作为寒冷和保护，防止肥胖2,3。这是通过解偶联线粒体解偶联蛋白1（UCP1）的​​电子传输。这种蛋白质被认为是一个标志性非颤抖性产热作用BAT 3。在近几年的几项研究显示，成年人有功能BAT 4-8，因此，在人类的BAT操作可能是一个潜在的干预治疗，对肥胖及其相关疾病的战斗中。 jove_content“目前的证据表明，棕色脂肪细胞中存在两种类型的啮齿动物，”古典“或”以前存在的“棕色脂肪在产前阶段的发展，形成专用的棕色脂肪在肩胛间区的仓库和其他外围组织。另一方面，“诱导”形式的棕色脂肪（所谓BRITE或米色细胞）在产后阶段发展，穿插在白色脂肪组织中出现，这两种类型的棕色脂肪细胞也分离由不同发育起源。虽然预先存在的棕色脂肪细胞从myoblastsic像MYF5前体的产生，诱导BRITE /米色WAT中穿插的细胞从的非MYF5谱系9,10产生，此外，这种细胞类型的调控途径可能是不同的从MYF5派生棕色可以激活慢性暴露于寒冷的环境和发展的米色细胞（ 即 “黑”的白色脂肪）脂肪11。β3-肾上腺素能受体激动剂或PPARγ激动剂在成人中12-14。浅米色/ BRITE细胞有可能是一个有前途的治疗目标操作的整体能源平衡，并有可能成为肥胖治疗的一部分，因此，重要的是要了解确切的分子机制和信号途径，其中环境因素控制的发展米色细胞。 了解的分子控制的白色脂肪的褐变， 在体外实验中是最适合作为发生，而异步的前体脂肪细胞的分化，它是很难检测细胞原位 15。虽然对脂肪细胞发展的研究迄今为止进行主要对3T3-L1，3T3-F442A或HIB1的细胞系，例如，这些细胞系出现米色细胞缺乏的分子签名。另一方面，从皮下WAT中分离的原代脂肪细胞是最有可能重述议事录S褐变的白色脂肪的细胞自主的方式。在这里，我们提供了一个协议，用于从脂肪组织的间质血管部分有效隔离和白色脂肪PPARγ激动剂诱导褐变。罗格列酮已被证明是一种非常有效的调解员在这些细胞中的褐变。正如先前所建议的16，这蜂窝系统可用于为可靠的蜂窝系统研究米色/布里特细胞的发展。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> Collaginaseð </td><td>罗氏公司</td><td> 11088874103 </td><td></td></tr><tr><td>中性蛋白酶II </td><td>罗氏公司</td><td> 04942078001 </td><td></td></tr><tr><td> 氯化钙 </td><td></td><td></td><td></td></tr><tr><td> DMEM培养基</td><td>费舍尔</td><td> 10017-CV </td><td>与2,5克/升葡萄糖和L-谷氨酰胺，而不丙酮酸钠</td></tr><tr><td>胰岛素</td><td></td><td></td><td></td></tr><tr><td> T3（3,3&#39;，5 - 三碘-L-甲状腺原氨酸） </td><td>西格玛</td><td> T-2877 </td><td></td></tr><tr><td>吲哚美辛</td><td>西格玛</td><td> I-7378 </td><td></td></tr><tr><td>地塞米松</td><td>西格玛</td><td> D-1756 </td><td></td></tr><tr><td> IBMX </td><td>西格玛</td><td> I-5879 </td><td></td></tr><tr><td>罗格列酮</td><td>西格玛</td><td> R-2408 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>胶原涂层菜</td><td> BD </td><td> 354450 </td><td> 10 CM平板</td></tr><tr><td> 70微米的过滤器</td><td> BD猎鹰</td><td> 352350 </td><td>细胞过滤网，70μm的尼龙1/EA </td></tr></tbody></table>

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

棕色脂肪细胞的能力脱开在线粒体呼吸链和消散化学能量作为热量。 UCP1阳性棕色脂肪细胞在白色脂肪组织（所谓的米色或BRITE细胞）的发展是高度由各种环境线索，如慢性暴露于寒冷的环境或PPARγ激动剂诱导，因此，这种细胞类型具有潜在的治疗靶点肥胖的治疗。虽然大多数永生脂肪细胞线可以不概括的“褐变”的白色脂肪中文化的过程，原代脂肪细胞分离出间质血管部分在皮下白色脂肪组织（WAT）提供一个可靠的蜂窝系统研究的分子控制的米色/ BRITE细胞发展。在这里，我们描述了一个协议，有效隔离的主要前体脂肪细胞和为文化的米色/布里特细胞的诱导分化。褐变效果可评估的棕色脂肪选择性标记的表达雇员再培训计划，如UCP1。

原代脂肪细胞褐变的可访问通过测量UCP1和其他棕色脂肪特异性或选择性由定量RT-PCR的基因的mRNA的表达。示于图1是腹股沟WAT-衍生的原代脂肪细胞中的基因表达数据。将细胞诱导分化，分别在存在两种不同剂量的罗格列酮，在50纳米和500纳米。 4小时收获前10μMforskolin的细胞治疗的一个子集。这将引起循环AMP（cAMP）的细胞和，包括UCP1的表达，激活热基因程序。 <p class="jo...

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

主要从小鼠白色脂肪组织中分离出的白色脂肪前体细胞可以分化成浅米色/ BRITE细胞。这里介绍的是一个可靠的细胞模型系统，研究的分子调控“黑”的白色脂肪。

米色/布里特细胞的间质血管细胞的分离和分化

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 次观看.  University of California, San Francisco. 主要从小鼠白色脂肪组织中分离出的白色脂肪前体细胞可以分化成浅米色/ BRITE细胞。这里介绍的是一个可靠的细胞模型系统，研究的分子调控“黑”的白色脂肪。

视频: 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.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

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

科研

生物学

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.

诱导上皮和分析间质转化

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.

隔离和鼠标主动脉内皮细胞原代培养

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.

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

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.

鼠冠状动脉血管平滑肌细胞的分离

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.

肾毒素显微注射斑马鱼到模型急性肾损伤

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.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

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.

成人小鼠心脏干细胞的分离、鉴定及分化

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.

恒河猴脂肪干细胞的分离、增殖和分化

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.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

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