这项研究是由美国国立卫生研究院的研究经费（DK090868和GM092930要德州仪器）的支持。有一个在笼中的雷帕霉素模拟待批专利。

1。转染质粒DNA <ol><li>加入质粒DNA，0.5微克膜栓联邦储备银行（以下简称为的LDR）和0.5微克FKBP-Tiam 1 mRNA（37.5微升DH 2 O荧光蛋白标记的T-细胞淋巴瘤侵袭转移诱导蛋白1：激活RAC）加入1μLFuGENE HD。 </li><li>涡，在室温下孵育20分钟。同时，进行步骤1.3-1.8。 </li><li>每8井室以及50μL聚-D-赖氨酸洗净。 </li><li>每口井的中心洗DH 2 O </li><li> Trypsinize 80-85％汇合NIH-3T3细胞，并稀释至10毫升培养基（含10％胎牛血清的DMEM培养液）。 </li><li>取出375μL悬浮，离心1750转每分钟为3分钟。 </li><li>抽吸介质，小心不要打扰沉淀。 </li><li>加入750μL培养液W / 10％FBS和重悬。 </li><li> DH 2的O / DNA / FuGENE HD解决方案，并加入细胞悬液轻轻混匀。 </li><li>新增250个每口井（细胞/ DNA的悬浮液将填补了3口井）。 </li><li>转染细胞在37°C和5％的CO 2为15-18小时。 </li></ol> 2。 CRB解决方案的制备<ol><li>雷帕霉素生物素加合物的合成笼（CRB）。见“CRB综合计划”的细节。简而言之，准备单独囚禁和生物素基。然后囚禁成分是共轭雷帕（LC实验室），该产品随后加上素成分的点击反应7负担CRB手段。 </li><li>准备的CRB 1μM的股票在DMSO溶液。 </li><li> 1mg的抗生物素蛋白溶解在250的PBS液0.5毫升的Eppendorf管中。 </li><li> CRB原液中加入1μL抗生物素蛋白溶解。混合反相几次。 </li><li>孵育15分钟，在室温下产生CRB抗生物素蛋白结合（CRB-）解决方案。 </li><li>排除未结合的小分子和净化CRB-A使用大小排斥列。 </li></ol> 3。在显微镜和亚细胞水平的光照<ol><li>血清挨饿12小时，在无血清培养液转染细胞。 </li><li>轻轻地吸了媒体的细胞，并加入250μL准备好的400毫微米CRB解决方案（从2.6步）。 </li><li>可视化蛋白质动力学，光治疗前，后生活的细胞在细胞内的决议，旋转盘显微镜（ 图2a）。紫外光照射UV LED灯（365纳米）与外延荧光光（ 图2b）由耦合光学实现。 </li><li> MetaMorph软件被用来捕捉每15秒转染细胞的荧光图像。 </li><li>通过使用紫外光激发荧光染料涂在玻璃板定义照明点的坐标和大小。大小可以可以采用不同的放大倍数的物镜，通过选择不同的核心大小的光纤不同。 </li><li>膜皱裂，照射紫外光后，决定在细胞周围的背景水平。下面的两个步骤是前本地化效果对比。 </li><li>在全球范围内提供365纳米，由汞灯光照射的细胞。 </li><li>解散开锁，从而在DMSO，然后在PBS（400毫微米）最后的原始霉素，并添加成像室解决方案，以促使全球影响。 </li></ol> 4。代表结果一个例子具有代表性的结果如图3所示。在蜂窝边缘附近的一个小区域的紫外光照射迅速诱导形成局部褶皱只有在和附近的照射面积。为了证实这的确是当地的活动，然后，我们在全球范围的紫外线照射细胞，或添加雷帕散装媒体，WHICH诱导遍布全球的膜皱褶形成。为定量分析，我们分为四个象限的靶细胞，并计算在每个象限根据当地的皱褶形成，以及根据各种条件，包括控制（图4）全球刺激后的频率。这是只有这种结合的紫外线照射，CRB-，并适当FKBP联邦储备银行的结构，引起当地皱裂。 <img alt="图1" src="/files/ftp_upload/3794/3794fig1.jpg" /> 图1。使用笼雷帕霉素抗生物素蛋白结合（CRB-A）的紫外光空间局限蛋白二聚体示意图。紫外线照射劈开雷帕霉素和生物素之间的连接器，导致释放的，化学dimerizer（HE-拉帕努伊 ，C40的-O-（2 -羟乙基） -雷帕霉素）和及其产品。然后释放dimerizer扩散进入细胞，并诱导二聚体之间FKBP的POI（兴趣蛋白）和血浆MEMBRANE联邦储备银行只在照射区域（蓝圈）附近停泊。没有紫外线的照射下，FKBP和联邦储备银行不联营公司（红十字会），因此不产生任何效果。 （版权所有：ACS2011）6 <img alt="图2" src="/files/ftp_upload/3794/3794fig2.jpg" /> 图2。定制的共焦荧光显微镜图片。 （一）正面共聚焦显微镜（Axiovert 200，蔡司倒置）。 （二）关闭，为计划免疫荧光和紫外线照射的耦合单元的顶视图。 <img alt="图3" src="/files/ftp_upload/3794/3794fig3.jpg" /> 图3。空间局限在转染NIH3T3细胞局部紫外线照射引起的皱褶形成。共聚焦荧光图像与YFP的标签FKBP​​-Tiam 1 mRNA（RAC激活）和LDR（膜锚联邦储备银行的）转染的NIH3T3成纤维细胞进行照射只限于在其边缘附近L区海关在15秒210秒开始，在CRB-共轭在散装媒体存在的365纳米的光。其次是这种治疗与汞全局照明灯1秒，1063秒和全球雷帕最后400毫微米除了1560秒。细胞发现几乎没有惹怒活动开始（一），但显示空间局限惹怒后局部照明（黄圈）（B）。后的细胞显示全局照明和雷帕霉素（CD）除了全球皱褶形成，表明当地皱褶形成，RAC激活空间局限造成的。比例尺：10微米。 （版权所有：ACS2011）6 <img alt="图4" src="/files/ftp_upload/3794/3794fig4.jpg" /> 图4象限的转染NIH3T3细胞中的皱褶形成的频率。当地紫外线照射与CRB-13细胞及以下全球紫外线照射。CRB- </s豪华型除了（无紫外线照射），局部紫外线照射或不亲和实施了13日，5日和6细胞，分别。没有紫外线照射，或局部紫外线照射或无抗生物素蛋白在散装媒体所观察到的细胞没有显示与CRB-怎幺形成。我们收集了从细胞与低背景皱褶形成的数据，但有雷帕霉素诱导的皱褶形成，我们每个实验后检查，此外，雷帕霉素（最后400毫微米）的结果确认为全球皱褶形成能力。这些结果表明，CRB-A和当地的紫外线照射是在当地皱褶细胞形成所必需的。 （版权所有：ACS2011）6 缩写： FKBP：FK506结合蛋白 联邦储备银行：FKBP-雷帕​​霉素结合蛋白 Tiam 1 mRNA：T细胞淋巴瘤侵袭转移诱导蛋白1（RAC环境基金） 的LDR：林恩-DSAG链接联邦储备银行（林恩：Lyn激酶N-末端11氨基酸） 拉帕：C40的- O - （2 -羟乙基） -雷帕霉素 CRB：笼与雷帕霉素生物素基 CRB-：CRB抗生物素蛋白结合

<ol>
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没有利益冲突的声明。

我们描述了一个技术，它采用一种新型的笼状化合物与FKBP联邦储备银行的异二聚体系统一起为了操纵Rho GTP酶的活性，在几秒钟的时间尺度上精确的亚细胞定位。 本办法有三个限制。首先，因为该方法的基础上，对防止或允许跨质膜dimerizer扩散的目标细胞的位置需要质膜或附近。可能使该化合物进入细胞，而直到光线照射诱导二聚体的的CRB化学结构的进一步优化。随着这种dim...

<table border="1"><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>雷帕霉素</td><td> tecoland </td><td> RAPA99 </td></tr><tr><td> FuGENE高清</td><td>罗氏公司</td><td> 04709691001 </td></tr><tr><td>抗生物素蛋白</td><td>西格玛</td><td> A9275-10MG </td></tr><tr><td>二甲基亚砜</td><td>西格玛</td><td> D2650-5X5ML </td></tr><tr><td>体积排阻柱</td><td> GE医疗集团</td><td>自旋捕集的G-25 </td></tr><tr><td>玻璃底8室</td><td> Thermo Scientific的</td><td> 12-565-47 </td></tr><tr><td>倒置荧光显微镜</td><td>蔡司</td><td> Axiovert200M </td></tr><tr><td>紫外LED光源</td><td>拉普光电电子有限公司</td><td> UVILED </td></tr><tr><td>共焦旋转盘</td><td>横河电机公司</td><td> CSU10 </td></tr><tr><td> CCD相机</td><td>滨松</td><td> ORCA的-ER </td></tr><tr><td> 100X目的镜头</td><td>蔡司</td><td>计划复消色差透镜</td></tr></tbody></table> CRB综合计划（版权ACS2011）6 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="计划1" /></a> 合成条件：（一）BnBr，钾2 CO 3，二甲基甲酰胺（二）（五），三氟乙酸（C）（D）丙炔-BR K 2 CO 3，二甲基甲酰胺，3 f.HNO甘油，猫。 PTSA，甲苯（F）硼氢化钠3 CN的四氯化钛 ，乙腈（G）TF 2 O的吡啶（H），甲醇盐酸，（我）LiAlH 4，四氢呋喃（J）敦沛证券，吡啶，CH 2 CL 2（K）为NaN 3，二甲基甲酰胺（L）8，2,6 -二叔丁基吡啶，CH 2 CL 2（米）13， 硫酸铜 ，抗坏血酸，2 -丙醇，H 2 O CH 2 CL 2。

spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells

Rho家族的小的鸟嘌呤triphosphatases与伟大的时空精度（GTP酶）的动态监管是必不可少的各种细胞功能和事件1，2。已经揭示了他们的活动可视化和实时3本地化他们spatiotemporally动态性质。为了获得更深入地了解自​​己的角色在不同的细胞功能，在分子水平上，下一步应该是摄蛋白质的活动在一个精确的亚细胞定位和定时。 为了实现这一目标，我们已经开发出一种光致，时空，时间控制的小GTP酶激活两种技术结合的方法：（1）雷帕霉素诱导FKBP联邦储备银行的异二聚体和（2）的雷帕照片囚禁的方法。与使用雷帕霉素介导的FKBP-FRB异二聚体，我们已经开发出一种快速诱导includi小GTP酶的激活或灭活方法吴RAC 4，Cdc42的4 4 RhoA蛋白和Ras 5，其中雷帕霉素诱导易位的的FKBP融合GTP酶，或他们的活化，在联邦储备银行锚定在细胞膜。与此异二聚体系统的耦合，我们还制定了雷帕霉素类似物的照片囚禁系统。图片笼状化合物是一种小分子，被称为作为一个囚禁组与photocleavable保护组抑制其活动。为了抑制异二聚体活性完全，我们设计了1笼中雷帕霉素，被拴1高分子这样认为，由此产生的大量复杂的可以不穿过细胞膜，导致1细胞6内的化学dimerizer几乎没有背景活动。我们计划图1所示系统。与这两个系统的结合，我们在本地招募了RAC激活质膜上几秒钟的时间表，并取得了在亚利光致RAC激活EL 6。

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

被描述为轻小GTPase活性的时空控制的方法。这种方法是基于FKBP-FRB雷帕霉素诱导的异二聚体和照片囚禁系统。优化的光照射，使时空，时间控制在亚细胞水平的小GTP酶激活。

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

spatio-temporal-manipulation-small-gtpase-activity-at-subcellular

Research

JoVE Journal

Bioengineering

10.8K 次观看.  Johns Hopkins University. 被描述为轻小GTPase活性的时空控制的方法。这种方法是基于FKBP-FRB雷帕霉素诱导的异二聚体和照片囚禁系统。优化的光照射，使时空，时间控制在亚细胞水平的小GTP酶激活。

视频: Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

科研

生物工程

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

氧微环境中的单个胰岛水平的定量和临时控制

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...