我们非常感谢来自韦恩州立大学（MJA），从美国的老龄化研究（SMV）基金会的赠款，并以独立职业过渡奖（R00EB007129）来自国家生物医学成像研究所和国家研究院​​生物的途径启动资金卫生（MJA）。

1。 Metalation使用LnCl 3盐<ol><li>溶解在水中的配体产生30-265 mM的解决方案。在这个视频配体2 - （4 - isothiocyanatobenzyl）-二乙三胺五乙酸（P - SCN - BN - DTPA的）浓度在73毫米。 </li><li>通过添加1个M NH 4 OH解决配体的pH值调整到5.5和7.0之间。在这段视频中，1米的NH 4 OH溶液0.2毫升。 </li><li>溶解于水1-2 LnCl 3等值5-1000 mM的浓度与生产的解决方案。在这个视频，EuCl 3和GdCl 3分别用浓度为111毫米。过量的金属经常被用来驱动metalation来完成，从而简化了净化。 </li><li>边搅拌边添加LnCl 3配体的解决方案解决方案。 </li><li>此外LnCl 3后，调整所产生的反应混合物的pH值在5.5和7.0之间，加入0.2 M NH 4 OH。共0.5毫升0.2 M NH 4 OH溶液中使用这个视频。如果您的配体中含有酸敏感的官能团，在这一步调整pH值多次。注意 - 如果的解决方案就显得太基本，任何敏感的基地功能组别，如异硫氰酸盐，将被渲染为共轭无法使用。 </li><li>监视器通过pH值测量的反应。反应完成后，当pH值保持不变。 </li></ol> 2。提高pH值workup（不包括在这个视频，但无基敏感官能团配体的良好） <ol><li>加浓氨水反应混合物中，调整pH值≥11 。这一步将不溶于水的氢氧化物沉淀为任何不拔的金属。 </li><li>通过0.2微米的过滤器的过滤上清。如果反应混合物堵塞过滤器，离心过滤前调迁建议。 </li><li>如果将不会被执行透析，除去溶剂，减少压力（旋转蒸发或建议冷冻干燥）下。 </li><li>如果仍然免费稀土，可重复步骤2.1-2.3。 </li></ol> 3。透析workup <ol><li>透析管切割到适当的长度（按照制造商的指引）举行的样本量，同时留出额外的长度（约10％的样本量）。在这段视频中，一个100-500道尔顿分子量（MWCO）切断膜，但如果接合之前，执行metalation较大的截留分子量管，可酌情使用。此外，透析磁带可用于透析管的替代品，如果需要的话。 </li><li>如果合适的基础上制造商的指引，在水中浸泡切透析管，在环境温度为15分钟。 </li><li>装满水（透析液）透析水库（1升烧杯中，在本视频中使用）。透析液量应约100倍的样品。 </li><li>折叠一端油管两次透析封钳油管安全的折叠部分。包装用橡皮筋封，以确保它仍然是封闭的透析过程中结束。 </li><li>反应混合物通过0.2微米的过滤器过滤，滤液装入小心不要撕破管材管的开口端。一定要留下足够的头部空间，关闭油管。 </li><li>油管剩余的开口端折两次，安全与封闭，封闭包装与步骤3.4橡皮筋。 </li><li>安装一个玻璃小瓶，含有空气透析管，使用橡皮筋的一端钳。将一小瓶含沙的其他钳。这些小瓶确保管子留在透析液中浸泡。 </li><li>将包含透析液透析水库管。 </li><li>搅拌透析液中，用磁力搅拌盘速度缓慢，在环境温度（无震荡）。 </li><li>变化的透析超过一天的课程（在此视频中，透析液改变为2.5，6.5，11.5 h）的3倍，然后让透析继续隔夜（20-28 h透析）。 </li><li>从透析液中取出的透析管，小心地打开一个封闭取出样品。洗净透析管水3倍，并与样品相结合的洗涤。 </li><li>卸下减压下的水。冷冻干燥是在这个视频。 </li></ol> 4。评估自由金属的存在<ol><li>金属配合物溶解在醋酸缓冲液（缓冲液配制：溶解在400毫升的水1.4毫升醋酸，调节pH至5.8，用1 M NH 4 OH，并加水产生总量为500 mL），并添加二甲酚橙指示剂在pH 5.8的缓冲（16微米）。在这个视频，0.3毫克复杂是0.3缓冲液溶解，并加入3毫升指示剂。 </li><li>检测自由金属通过观察一个指标的颜色从黄色紫色变化。 </li><li>如果需要，可用金属量可量化的创建校准曲线1。另外，染料偶氮胂III可以用来代替二甲酚橙2。如果免费的金属仍然存在，样品应进一步纯化，使用前透析，脱盐柱，高效液相色谱（HPLC）来表征。 </li></ol> 5。测定水协调的数量（Q ） <ol><li>准备一个解决方案，欧盟三复杂的含H 2 O（〜1毫米），另一个在D 2 O的浓度相同的解决方案分析之前，必须在D 2 O解决方案蒸干，溶解在D 2 O 3次，以去除残留的H 2 O </li><li>水溶液加入到一个干净的比色皿，试管放入一个分光光度计。 </li><li>执行的激发和发射扫描，以确定每个（〜395 nm和〜595纳米，分别）千里马。 </li><li>执行一个磷光时间衰变实验，使用下列参数：激发和发射波长从初始延迟（0.01毫秒），步骤5.3中，激发和发射狭缝宽度（5纳米），闪光次数（100），最大延迟时间（13毫秒），确定和延迟增量（0.1毫秒）。这些条件是合适的，但最络合物的最大延迟和增量值可以增加或极长或极短的衰减时间下降的物种。 </li><li>重复步骤5.4步骤5.1准备与D 2 O解决方案。 </li><li>从5.4和5.5获得的发光衰减数据，情节强度随时间变化的的自然对数。这些线的斜率衰减率（τ- 1）（图2） 。在这段视频中，Microsoft Excel 2007中使用生成的原始数据的自然对数图。霍罗克斯和他的同事（EQ）3开发的公式中使用的衰变率。如果您的配体中含有OH或NH组协调，以金属，则方程必须修改使用前3。 </li></ol> 公式1： <img src="/files/ftp_upload/2844/2844eq1.jpg" alt="公式1" /> 6。弛豫测量<ol><li>选择弛豫时间分析仪所需的应用模式 ：T 1（纵向弛豫时间） 或 T 2（横向弛豫时间）。 </li><li>准备了一系列的样品含有不同浓度的含钆三 ，在水溶剂的复杂。在这段视频中，水被用作溶剂和解决方案为10.0，5.00，2.50，1.25，0.625，0毫米准备。其他水溶剂或缓冲区可以使用，但重要的是要使用的溶剂为空白。样品的最终体积是针对正在使用的仪器。 </li><li>样品放置在仪器，让它坐5分钟，以平衡仪器的温度（37℃，在此视频）。 </li><li>通过调整软件的参数，以获得平滑指数曲线为 T 1 或 T 2（代 ​​表为 T 1 和 T 2曲线如图3所示），确定弛豫时间（s为单位） 。 </li><li>所有样本包括空白，重复步骤6.3和6.4。 </li><li>计算测 T 1 或 T 2值，单位为s - 1时的倒数。 </li><li>绘制T 1 -1 或 T 2 -1与GD 第三浓度值（单位为mm）。由于III含钆复合物的吸湿性质， 确认第三钆的浓度，用原子吸收光谱法或电感耦合等离子体质谱。拟合直线的情节。代表图如图4所示。 </li><li>拟合直线的斜率是弛豫（T 1 和 T 2分别为R 1 或 R 2）和MM -1 s - 1时的单位。 </li></ol> 7。代表性的成果已包括在这个协议的步骤有代表性的数据表和数字部分。除了在协议中所描述的情况下，协调水的数量和弛豫特性，重要的是要使用标准的化学技术的最终产品的特点。该化合物的身份可以得到使用，和有代表性的质谱质谱显示钆三同位素诊断模式- 欧盟III -含有复合物，如图5所示。此外，非钆三 </s&gt;含稀土配合物，核磁共振光谱可用于产品标识。为了描述复杂的纯度，高效液相色谱，元素分析，或两者都可以使用。 <img src="/files/ftp_upload/2844/2844fig1.jpg" alt="图1" /> 图1总体方案metalation和净化：计划描绘的一般程序metalation和用于选择不同的净化路线的原因。 <img src="/files/ftp_upload/2844/2844fig2.jpg" alt="图2" /> 图2发光强度的情节：代表的强度与时间从第5的自然对数的情节。用于生成水和D 2 O解决方案获得类似的曲线线条的斜坡式1来表征水配位数的欧盟III含络合物。 <img src="/files/ftp_upload/2844/2844fig3.jpg" alt="图3" /> 图3：松弛的衰减时间曲线：（左）T 1和（右）T 2采集有代表性的数据。从这些曲线形状的偏差，会产生不可靠的数据。 <img src="/files/ftp_upload/2844/2844fig4.jpg" alt="图4" /> 图4弛豫决心：一个有代表性的情节与浓度钆三 ：1 / T 1。拟合直线的斜率是弛豫和MM -1 s - 1时的单位。 <img src="/files/ftp_upload/2844/2844fig5.jpg" alt="图5" /> 图5质谱：代表质谱诊断同位素模式（左）钆三含复合物和（右）欧盟III含有复合物。黑色的高斯峰代表理论同位素分布，红色线是实际的数据。

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鉴于越来越多的出版物，包括镧系元素的造影剂4-14，这是重要的准备，净化和表征产品，以确保重现性好，比较的结果，采取照顾。这些复合物通常被认为是具有挑战性的净化和表征有机分子由于其顺磁性质和任何可以使用bioconjugation的功能组别的敏感性相对。我们已经描述了稀土配合物的合成，纯化，并表征的常用方法。然而，选择其中一种方法时，重要的是要考虑的具体制度，正在?...

<table border="1"><tbody><tr><th>试剂和仪器</th><th>公司</th><th>目录编号</th></tr><tr><td> EuCl 3•6H 2 O </td><td> Sigma - Aldrich公司</td><td> 203254 - 5G </td></tr><tr><td> P - SCN - BN - DTPA </td><td> Macrocyclics </td><td> B - 305 </td></tr><tr><td>氢氧化铵</td><td>电解二氧化锰</td><td> AX1303 - 3 </td></tr><tr><td>光谱/ POR生物纤维素酯（CE）的透析膜 - 500 ð截留分子量</td><td> Fisher Scientific则</td><td> 68-671-24 </td></tr><tr><td> Millipore公司IC Millex - LG过滤单元</td><td> Fisher Scientific则</td><td> SLLG C13 NL </td></tr><tr><td>二甲酚橙四钠盐</td><td>阿法埃莎</td><td> 41379 </td></tr><tr><td>乙酸</td><td> Fluka公司</td><td> 49199 </td></tr><tr><td> D 2 O </td><td>剑桥同位素实验室公司</td><td> DLM - 4 - 25 </td></tr><tr><td>净水器</td><td> ELGA </td><td> Purelab超</td></tr><tr><td>高效液相色谱法和质谱</td><td>日本岛津</td><td> LCMS - 2010EV </td></tr><tr><td>弛豫时间分析仪</td><td>布鲁克</td><td> mq60 minispec </td></tr><tr><td>紫外可见分光光度计</td><td> Fisher Scientific则</td><td> 20-624-00092 </td></tr><tr><td>冷冻干燥机</td><td> Fisher Scientific则</td><td> 10-030-133 </td></tr><tr><td>酸度计</td><td> HANNA INSTRUMENTS </td><td>您好221 </td></tr><tr><td>分光光度计</td><td> HORIBA Jobin Yvon公司</td><td> Fluoromax - 4 </td></tr><tr><td>分子量计算器马修梦露6.46版本，下载2009年10月17日，日</td><td> <a href="http://ncrr.pnl.gov/software/">http://ncrr.pnl.gov/software/</a> </td><td>分子量计算器</td></tr></tbody></table>

preparation, purification, and characterization of lanthanide complexes for use as contrast agents for magnetic resonance imaging

常用Polyaminopolycarboxylate基于配体螯合稀土离子，并由此产生的复合物作为磁共振成像（MRI）造影剂。许多市售的配体是特别有用，因为它们包含的功能组别，允许快速，高纯度和高产通过胺反应活性酯和异硫氰酸团体或巯基反应马来酰亚胺的大分子和生物分子共轭。虽然这些配体metalation bioconjugation化学细微的差别，在metalation程序方面的常识，必须考虑选择金属起始原料时。此外，纯化及鉴定的存在，并选择最有效的程序的多个选项部分取决于起始材料的选择。这些细微的差别往往被忽视发布的协议。在这里，我们的目标是展示metalation，纯化和特​​性，可作为MRI（图1）造影剂使用的稀土配合物的常用方法。我们期望本出版物将使生物医学科学家将其纳入自己的常用反应剧目稀土络合反应，由宽松的起始材料和净化方法的选择。

Watch this Scientific Journal Video about Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging at JoVE.com

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

我们展示metalation，净化，稀土配合物的特性。这里所描述的复合物，可以结合大分子，使这些分子利用磁共振成像进行跟踪。

制备，纯化和磁共振成像造影剂的使用稀土配合物的表征

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

preparation-purification-characterization-lanthanide-complexes-for

Research

JoVE Journal

Biology

14.9K 次观看.  Wayne State University. 我们展示metalation，净化，稀土配合物的特性。这里所描述的复合物，可以结合大分子，使这些分子利用磁共振成像进行跟踪。

视频: Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

科研

生物学

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

在人类胚胎干细胞体外培养的标签用于磁共振成像

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

相衬，微分干涉对比（DIC）显微镜

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

作为一个型号的遗传生物鳉胚胎显微注射

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

量化混合使用磁共振成像

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

产生和人类INO80染色质重塑复合物及其亚纯化

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

双正电子发射层析成像用 68 ga 芯掺杂氧化铁纳米粒子的合成/(t1) 磁共振成像

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...