我们感谢Meshorer实验室的成员，尤其是晒Melcer，阿迪Alajem，Edupuganti Raghu RAM，巴迪斯里兰卡Sailaja，安娜Mattout和阿尔瓦必然的批评和故障排除漂白实验每天。 EM是约瑟夫H和Belle R.布朗在生命科学学院的高级讲师，是由以色列科学基金会（ISF 943/09），以色列卫生部（6007）欧洲联盟（IRG - 206872和238176）的支持，以色列癌症研究基金会，内部应用性的希伯来大学和以色列精神生物学研究所的医疗补助金。

1。电镀ES细胞 T = 0小时 MEF的电镀<ol><li>大衣实时成像8以及μ-幻灯片（德国ibidi;慕尼黑），明胶（实验室TEK;罗切斯特，纽约州）在墓室盖眼镜或玻璃底培养皿（MatTek;阿什兰，马萨诸塞州）。离开5-30分钟，吸了免费的明胶。 </li><li> 22000种MEFs / 250μLDMEM总量10％胎牛血清（FBS），补充]。让细胞生长在一个组织培养箱培养（37℃，5％CO2）。 </li></ol> T = 6小时 ES细胞的电镀<ol start="3"><li>吸的DMEM。 </li><li>种子/ ES细胞介质的总体积250μL涂15000 R1的细胞以及每个MEF [辅以10％ESC级小牛血清（FBS），丙酮酸钠1毫米，0.1毫米非必需氨基酸，0.1毫米的β-巯基乙醇，和1000 U / ml的白血病抑制因子（LIF）]，以获得30％至50％汇合翌日。 </li></ol> 2。转染ES细胞 T = 24小时 瞬时转染<ol><li>替换ES细胞介质与250μL/孔新鲜胚胎干细胞的媒体。 </li><li>在1.5毫升的无菌试管中，加100μL无血清介质[OPTI - MEM（GIBCO）]，然后直接添加到无血清媒体10μL过境LT1转染试剂（Mirus）。轻轻吹打混合，在室温下孵育5-20分钟。 </li><li>添加1.5微克GFP融合的质粒DNA（H1e，H1o或HP1）稀释过境LT1试剂。轻柔吹打混匀，室温孵育15-30分钟。 </li><li>加入13.5μL/孔的转染混合物。 8漩涡以及μ-幻灯片，以确保即使疏散。经过24小时的250μL新鲜的胚胎干细胞媒体取代旧的ES细胞的媒体。 </li></ol> 3。执行FRAP和FLIP T = 48-72小时<ol><li>实验可以在任何激光扫描共聚焦显微镜（CLSM），但因为在一个正常的FRAP /翻盖实验，获得了许多连续的影像，这是推荐使用一个旋转的磁盘共聚焦显微镜，使采集速度，并确保没有不受欢迎的样品漂白会发生以下初始故意漂白事件。在这里，我们推荐使用旋转盘共聚焦系统（www.Andor.com）的革命，与横河电机CSU - X的旋转盘头。该系统具有光漂白点扫描系统使用一个专门的FRAPPA模块的双重身份，和快速切换的光背，采集图像，使用旋转盘。用于漂白实验的3个最常见的荧光蛋白质GFP，YFP和樱桃。如果使用GFP或YFP，一个〜488 nm激光是必需的。樱花，使用一个〜560纳米的激光。在所有情况下，我们建议使用固态激光器。一个自动化的阶段，是非常有用的，但不是必需的。由于活细胞成像，它是必不可少的使用环境室（我们使用LIS之一，瑞士），控制氧气，湿度，CO 2和温度。 FRAP是使用最大的激光强度，而成像所需的最小激光功率（通常在10％的面积，当荧光水平是足够的）。 细胞，并用适当波长的荧光光观察，选择一个单元格，使用60X油浸镜头表达GFP。确保正确的亚细胞分布。有时，表达水平过高时，蛋白质的本地化可能“外溢”到其他车厢，如核仁。这种细胞不应该被选中。 </li><li>现在设置的成像协议：收集的漂白前3-5帧，然后在常染色质或异（简明GFP的疫源地浏览）的光漂白和收集的光漂白后90-120帧250-1000毫秒的时间间隔，H1e - GFP，1000 MS，H1o和HP1 - GFP，250毫秒（根据蛋白质动力学，极具活力的蛋白质，需要较短的时间间隔时间间隔时间的变化）。我们通常使用漂白用20-40μseconds（1-2迭代）的激光脉冲的80-100％的激光强度，但据分析蛋白质的表达水平，这些数字可以改变。当漂白是合适的，你应该遵守一个“黑洞”在你的绿色荧光。黑洞是逐步重新充满荧光以下恢复。虽然旋转盘，可取得最多约每秒60图像（具有良好的荧光强度和单个细胞在缩放时），我们不建议使用在这样低的图像质量和增加光毒性潜力的高速系统。 </li><li>对于一个翻转实验，设置了不同的的成像协议：收集前漂白3-5帧，然后开始反复漂白，而在同一地点采集图像。 H1e - GFP H1o - GFP，漂白剂，漂白剂，每隔5秒，每2秒，HP1 - GFP漂白每1秒。死神一个ð重复采集图像，整个实验。 </li><li>对于这两种技术，重复20-30细胞的过程。用于统计目的的，重复实验3次或以上，最好是在不同的日子。在同质人口和正确的设置，标准偏差通常较低（&lt;5％）。 对于这两个FRAP和FLIP，漂白区域的大小和形状会影响经济复苏动力，必须保持在一个实验常数。此外，当两个单元格进行比较，相同的协议必须使用和细胞必须按顺序在同一天功率激光和其他条件可能出现波动，可能会影响实验的结果进行分析。 </li></ol> 4。 FRAP和分析翻转数据<ol><li>在所有FRAP帧收集，测量的荧光强度，投资回报率（ROI b =漂白区），背景区域（BG的投资回报率），和非漂白的核面积作为时间的函数之前，漂白后（投资回报率，NB） 。当漂白地区是微不足道的，整个核可以选择正常化的目的。 </li><li>对于每一个时间点，正常化数据根据公式：（投资回报率B -投资回报率BG）/（NB的投资回报率-投资回报率BG）/（pbROI B - pbROI BG）/（pbROI NB - pbROI BG），PB表示预漂白。漂白前的图像，你应该得到的价值约1。漂白后的第一个图像将显示漂白的深度。减去实际漂白深度值的值从1。重复每一个细胞，每个实验的平均20-30细胞。 </li><li>在收集到的所有翻转帧，测量荧光强度，在非漂白核领域和背景区（NB =非漂白领域的投资回报率，投资回报率BG =背景）。翻盖数据的计算是类似一个FRAP曲线，只有分析的ROI（投资回报率 NB）应比实际漂白的地区，这是不用于计算不同 ：（NB的投资回报率- 投资回报率BG）/（pbROI NB - pbROI BG） 。正常化的目的，它也可以使用邻近细胞（投资回报率N =相邻小区）：NB -投资回报率（ROI BG）/（投资回报率N -投资回报率BG）/（pbROI NB - pbROI BG）/（pbROI N - pbROI BG ）。 以下数据收集，这是可能的，以适应计算机模拟实验数据。这允许计算，具有良好的接近，移动的一小部分，动弹不得的分数和半最大，。我们不会讨论FRAP分析的数学和统计方面和读者参考其他优秀出版物4-9。漂白深度是指预漂白剂（100％）的信号和漂白后的第一形象之间的距离（Y轴）移动的分数是指之间的漂白剂的深度和距离（y轴）恢复的信号时的动力学到达高原，不动的一小部分，是指恢复的信号和预漂白剂（100％）信号之间的距离（Y轴）（见图1B和2B）。除了这一分析，有良好的数学模型来拟合数据。对于一个单一的指数，该方程 <img src="/files/ftp_upload/2696/eq0.jpg" alt="方程" width="80" /> 其中t是时间，一个是移动的一小部分，1，一个是不动的分数和K过的解离常数，可用于数据拟合，并可以直接估计一个有约束力的帐率（K关闭 ）获得，以及参数，可用于计算的关联率。 </li></ol> 5。代表性的成果： <img src="/files/ftp_upload/2696/2696fig1.jpg" alt="图1" /> 图1：A和B显示代表HP1（左），H1o（中）和H1e（右）在R1 ES细胞的FRAP曲线。对于简单和清晰图1A显示任何正常化和计算前的一个单细胞的原始数据。黄色曲线对应的漂白地区，紫色的曲线对应的非漂白的核区（漂白地区是微不足道的，整个核可以正常化的目的选择），红线对应的背景荧光，这是在这种情况下最小。垂直箭头代表漂白的时间。规范化和平均数据显示在图1B。注意H1（蓝色）HP1（红色）相比，恢复较慢。也H1e变种（深蓝色）是比H1o变种（浅蓝色）慢。移动和静止的分数和漂白剂深度为HP1表示。 <img src="/files/ftp_upload/2696/2696fig2.jpg" alt="图2" /> 图2，A和B显示代表FRAP曲线比较常染色质（左异的HP1（右）在R1 ES细胞）。同样以图1，图2a显示了一个单细胞的原始数据，黄色曲线对应的漂白地区，紫色曲线对应的非漂白的核面积，红线对应的背景荧光。垂直箭头代表漂白的时间。规范化和平均数据，如图2b所示。注意异（暗红色）与常染色质（淡红色）相比，恢复较慢。移动和静止的分数和漂白剂深度表示为常染色质。 <img src="/files/ftp_upload/2696/2696fig3.jpg" alt="图3" /> 图3。一个典型的H1o R1 ES细胞的另一实验显示在图3A（原材料，联合国规范化的数据）和B（正常化和平均数据）。在这个实验中，紫色的曲线对应的非漂白的核领域，绿色线对应到邻近的细胞的细胞核和红线对应背景荧光。

<ol>
	<li>Phair, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+nature+of+dynamic+protein-chromatin+interactions+in+vivo:+three-dimensional+genome+scanning+and+dynamic+interaction+networks+of+chromatin+proteins.">Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins.</a> Mol Cell Biol. 24, 6393-6402 (2004).</li><li>Meshorer, E., Girard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging chromatin in embyonic stem cells in StemBook. , (2008).</li><li>Meshorer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperdynamic+plasticity+of+chromatin+proteins+in+pluripotent+embryonic+stem+cells.">Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells.</a> Dev Cell. 10, 105-116 (2006).</li><li>Bancaud, A., Huet, S., Rabut, G., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, photoactivation, photo conversion, and FLIP. , (2009).</li><li>Dundr, M., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+dynamics+of+nuclear+proteins+by+photobleaching.">Measuring dynamics of nuclear proteins by photobleaching.</a> Curr Protoc Cell Biol. Chapter 13, Unit 13-Unit 13 (2003).</li><li>Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+membrane+dynamics+and+reassembly+in+living+cells:+targeting+of+an+inner+nuclear+membrane+protein+in+interphase+and+mitosis.">Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis.</a> J Cell Biol. 138, 1193-1206 (1997).</li><li>Lenser, T., Weisshart, K., Ulbricht, T., Klement, K., Hemmerich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+fluctuation+microscopy+to+reveal+3D+architecture+and+function+in+the+cell+nucleus.">Fluorescence fluctuation microscopy to reveal 3D architecture and function in the cell nucleus.</a> Methods Cell Biol. 98, 2-33 (2010).</li><li>Mueller, F., Mazza, D., Stasevich, T. J., McNally, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+and+kinetic+modeling+in+the+analysis+of+nuclear+protein+dynamics:+what+do+we+really+know.">FRAP and kinetic modeling in the analysis of nuclear protein dynamics: what do we really know.</a> Curr Opin Cell Biol. 22, 403-411 (2010).</li><li>Phair, R. D., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+modelling+approaches+to+in+vivo+imaging.">Kinetic modelling approaches to in vivo imaging.</a> Nat Rev Mol Cell Biol. 2, 898-907 (2001).</li><li>Poser, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+TransgeneOmics:+a+high-throughput+method+for+exploration+of+protein+function+in+mammals.">BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.</a> Nat Methods. 5, 409-415 (2008).</li><li>Sigal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+fluorescently+labeled+endogenous+protein+library+in+living+human+cells.">Generation of a fluorescently labeled endogenous protein library in living human cells.</a> Nat Protoc. 2, 1515-1527 (2007).</li><li>Cohen, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+proteomics+of+individual+cancer+cells+in+response+to+a+drug.">Dynamic proteomics of individual cancer cells in response to a drug.</a> Science. 322, 1511-1516 (2008).</li></ol>

与大多数可用的技术，涉及从细胞群或固定细胞，FRAP实验纯化染色按照在活细胞中染色质的蛋白质动力学变化。我们发现染色质蛋白的动态染色质可塑性的一个良好指标。然而，因为它需要融合的兴趣与绿色荧光蛋白基因，荧光标记除了可以干扰蛋白质的功能。因此，与FRAP出发之前，该融合蛋白，必须经过严格的测试，以确保它具有相同的属性和作为其原生对应的功能。金标准将在淘汰赛细胞?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> DMEM培养液</td><td>西格玛</td><td> D5671 </td></tr><tr><td>明胶</td><td>默克公司</td><td> 1.04078 </td></tr><tr><td> OPTI - MEM </td><td> Gibco公司</td><td> 31985 </td></tr><tr><td>中转- LT1 </td><td> Mirus </td><td> MIR2300 </td></tr><tr><td> 8以及μ-幻灯片</td><td> ibidi </td><td> 80826 </td></tr></tbody></table>

photobleaching assays (frap &amp; flip) to measure chromatin protein dynamics in living embryonic stem cells

漂白后荧光恢复（FRAP）和漂白荧光丢失（FLIP），使具有良好的空间和时间分辨率的活细胞中的蛋白质动力学研究。在这里，我们描述了如何执行FRAP翻转检测和染色质蛋白，其中包括H1和HP1，在小鼠胚胎干细胞（ES）。在一个FRAP实验中，细胞转染，无论是瞬时或稳定，与一个融合绿色荧光蛋白（GFP）及其衍生物（YFP，CFP，樱桃等）的利益的蛋白质。在转染，荧光的细胞，强烈聚焦的激光束漂白剂的利益相对较小的区域（ROI）。选择的激光波长是根据荧光蛋白融合使用。激光光的投资回报率不可逆的漂白剂分子的荧光信号后，立即漂白，漂白面积的荧光信号的恢复 - 更换漂白漂白分子的分子介导的 - 监测使用时间的推移成像。产生的荧光恢复曲线对蛋白质的流动性提供了信息。如果荧光分子不动，无荧光的复苏将是观察。互补的方法，在漂白荧光丢失（FLIP），激光束的漂白剂在同一地点反复和信号强度测量荧光细胞。因此，翻转实验测量信号衰减而不是荧光恢复，并有助于确定蛋白质的流动性以及蛋白质，细胞车厢之间穿梭。瞬态约束力的染色质相关蛋白的共同财产。虽然每个染色质蛋白的主要部分是在任何给定时刻处于稳定状态的约束染色，绑定是暂时性的，最染色质蛋白对染色质高价成交，停留时间在几秒钟的顺序。这些属性1基因的表达产生的高可塑性的关键。漂白实验，因此特别有用，以确定染色质的可塑性，尤其是在胚胎干细胞，使用的染色质结构蛋白GFP融合版本是较高的染色质蛋白（包括异染色质蛋白1（HP1），连接器的组蛋白H1和核心组蛋白）的动态交换比2,3分化的细胞。

Watch this Scientific Journal Video about Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells at JoVE.com

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

我们描述漂白方法，包括荧光漂白后恢复（FRAP）和荧光漂白（翻转）监测胚胎干细胞（ES细胞）的染色质的蛋白质动力学的损失。染色质蛋白的动态，这被认为是研究染色质可塑性的手段之一，是增强多能干细胞。

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

photobleaching-assays-frap-flip-to-measure-chromatin-protein-dynamics

Research

JoVE Journal

Biology

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

27.0K 次观看.  The Hebrew University of Jerusalem. 我们描述漂白方法，包括荧光漂白后恢复（FRAP）和荧光漂白（翻转）监测胚胎干细胞（ES细胞）的染色质的蛋白质动力学的损失。染色质蛋白的动态，这被认为是研究染色质可塑性的手段之一，是增强多能干细胞。

视频: Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

图案的使用生物倒装芯片的胚胎干细胞

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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 ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

使用plusTipTracker软件测量微管动力学<em&gt;非洲爪蟾</em&gt;生长锥

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...