这个项目是一个莱昂J.塔尔从加州再生医学研究所的种子批。托比亚斯亨宁是由德国研究协会（DFG，何4578/1-2）津贴一个研究。我们要感谢他对人类胚胎干细胞培养的意见的华尼托Meneses。

标识与Ferucarbotran HMSC 这是标签与Ferucarbotran，氧化铁磁共振成像造影剂人类间质干细胞的技术： <ol><li>首先，板18-24小时前标签程序在T75烧瓶在80％汇合的细胞。如果您使用一个不同的培养皿中，这是相等于约10000细胞每平方厘米 2 。 </li><li>第二天，开始准备的标签介质，加入30μLResovist无血清媒体8毫升。将标签之一T75烧瓶中，在80％汇合对应一个铁100微克/毫升媒体的浓度。 </li><li>起飞培养基，用PBS或无血清培养基洗细胞一次。这样做是为了清除残留的血清蛋白质和其他成分的媒体，可以结合造影剂和影响标记效率。 </li><li>添加标签媒体烧瓶中，并把在孵化器的烧瓶回。 </li><li> 2小时后，加入使终浓度达到20％FCS的FCS2毫升。这样做是为了确保这些细胞没有收到刺激分化，而是在自己熟悉的环境中成长。 </li><li>细胞培养超过18小时。这是最好的一蹴而就。 </li><li>第二天，用PBS冲洗细胞，然后trypsinize他们像往常一样。 </li><li>胰酶消化后，细胞悬液，需要洗3次，在媒体的残留，无造影剂摆脱。这是用PBS离心5分钟，在400 RCF细胞。 </li><li>最后细胞沉淀重悬，并准备作进一步的实验。 </li><li>现在，因为有些人可能已经在过去的洗涤步骤失去的细胞计数。此外，执行使用台盼蓝排斥实验点的可行性测试，并采取一些样品光谱分析测量标记效率。 </li></ol> 人类胚胎干细胞的标记与 Ferumoxides <ol><li>首先，板块10厘米菜的胚胎干细胞，像往常一样。这些菜是用明胶预涂，对他们的辐照饲养层细胞。您可以使用免费馈线文化，以及本议定书。 </li><li>让我们的人类胚胎干细胞附着和生长约3-4天，使他们形成中等大小的菌落。在这段时间内，确保殖民地不长到大，更大的殖民地是更难以打破后的。 </li><li>分化的细胞污染，使这些文化殖民地的清洁度而定，您可能已经摆脱夹层有区别的文化。 </li><li>准备，由浓度100μg/ml和完整的人类胚胎干细胞媒体Ferumoxides标签媒体。对于这一点，我们的混合89μlFerumoxides全面增长媒体和10毫升。 </li><li>洗净的菜，一次完整的媒体。 </li><li>添加10毫升每盘媒体的标签，并培育了4个小时的细胞。 </li><li>现在，标签是完整的。在接下来的步骤，它是要洗的细胞，并得到进一步利用单细胞悬液。对于这一点，首先用PBS冲洗菜。 </li><li>然后，替换5毫升0.25％胰蛋白酶的PBS，并在孵化器中孵化5分钟，或直至细胞分离。它有助于挖掘的菜经常。 </li><li>细胞分离。请记住，如果菜有较大的殖民地可能会有一些团块离开。 </li><li>为了摆脱这些团块，可以混合一次使用血清学吸管整个悬浮液，然后让它坐在另2分钟。 </li><li>我们不使用的Eppendorf移液器，透过薄薄的尖端细胞反复吸吮可以打破细胞膜。 </li><li>如果剩余的团块，可以通过一个40微米的细胞过滤器运行的细胞悬液。 </li><li>如果一切工作正常，单细胞悬液，现在存在着大量泥石混合，明胶涂层的ESC矩阵和死细胞。为了摆脱这个，洗细胞两次5分钟旋转400 RCF重悬在全媒体。 </li><li>现在，人事编制小组委员会需要从辐照饲养层细胞中分离。这是可以做到有效的明胶涂层盘上镀细胞悬液。 </li><li>如果菜是不是操纵，所有细胞将泥沙下来，但进料器将附加速度比ESC。 </li><li>所以，如果45分钟后上清液，它应该包含主要是ESC键，而进料器主要应附菜。 </li><li>这样，一个磁标记的人类胚胎干细胞，可用于进一步的实验中使用的单细胞悬液。 </li><li>由于在以前的协议，在这一点上，你需要计数细胞标记效率的可行性评估和测量，并采取样品。 </li></ol>

<ol>
	<li>Daldrup-Link, H. E., Rudelius, M., Oostendorp, R. A. J., Settles, M., Piontek, G., Metz, S., Heinzmann, U., Rummeny, E. J., Schlegel, J., Link, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+Hematopoietic+Progenitor+Cells+with+MR+Contrast+Agents.">Targeting of Hematopoietic Progenitor Cells with MR Contrast Agents.</a> 228, 760-767 (2003).</li><li>Daldrup-Link, H. E., Rudelius, M., Piontek, G., Metz, S., Brauer, R., Debus, G., Corot, C., Schlegel, J., Link, T. M., Peschel, C., Rummeny, E. J., Oostendorp, R. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+iron+oxide+labeled+human+hematopoietic+progenitor+cells+in+a+xenotransplant+model:+in+vivo+monitoring+using+clinical+magnetic+resonance+imaging+equipment.">Migration of iron oxide labeled human hematopoietic progenitor cells in a xenotransplant model: in vivo monitoring using clinical magnetic resonance imaging equipment.</a> Radiology. 234, 197-205 (2005).</li><li>Henning, T. D., Saborowski, O., Golovko, D., Boddington, S., Bauer, J. S., Fu, Y., Meier, R., Pietsch, H., Sennino, B., McDonald, D. M., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+labeling+with+the+positive+MR+contrast+agent.">Cell labeling with the positive MR contrast agent.</a> Gadofluorine M. Eur Radiol. 17, 1226-1234 (2007).</li></ol>

一个干细胞的非侵入性的描述是监测几乎所有的干细胞为基础的治疗是至关重要的。一个更好的理解，在标记的干细胞的体内动力学MR图像的可视化，可能导致理性的方式和更有效地利用干细胞疗法。由于我们使用了临床应用的造影剂和成像技术，我们提出的协议应随时翻译患者应用。潜在的临床应用我们的方法包括比较调查的各种亚型干细胞或基因改造的干细胞植入过程，以及评估结果的干细胞植入的治疗效?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>D-MEM High Glucose</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D5648</td>
<td>&nbsp;</td>
<tr>
<td>Gelatin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G1890</td>
<td>dilute Gelatin in PBS to final concentration of 0.1 %</td>
</tr>
<tr>
<td>PBS (Ca++ Mg++ freed)</td>
<td>Reagent</td>
<td>GIBCO</td>
<td> 14190-144</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA 0.05%</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25300-120</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA 0.25%</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25200-114	</td>
<td>&nbsp;</td>
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<td>FBS characterized</td>
<td>Reagent</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
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<td>Cell Strainer (40 &#x03BC;m Nylon)</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
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<td>Knockout DMEM</td>
<td>Reagent</td>
<td>GIBCO</td>
<td>10829018</td>
<td>&nbsp;</td>
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<td>Knockout Serum Replacer</td>
<td>Reagent</td>
<td>GIBCO</td>
<td>10828028</td>
<td>&nbsp;</td>
<tr>
<td>b-Mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>7522</td>
<td>&nbsp;</td>
<tr>
<td>non-essential amino acids</td>
<td>Reagent</td>
<td>GIBCO</td>
<td>11140050</td>
<td>&nbsp;</td>
<tr>
<td>FGF-2</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>233-FB-025</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>GIBCO</td>
<td>25030081</td>
<td>&nbsp;</td>
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<td>Penicillin/Streptomycin</td>
<td>Reagent</td>
<td>GIBCO</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Ferumoxides (Endorem)</td>
<td>Reagent</td>
<td>Guerbet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ferucarbotran (Resovist&#174;)</td>
<td>Reagent</td>
<td>Bayer Schering AG</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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labeling hescs and hmscs with iron oxide nanoparticles for non-invasive in vivo tracking with mr imaging

近年来，干细胞研究导致了一个更好地理解，发育生物学，各种疾病和再生医学上的潜在影响。一种非侵入性的方法来监视移植的干细胞在体内反复将大大提高我们的理解能力的机制，控制干细胞死亡，并确定营养因子及信号转导通路，提高干细胞植入。磁共振成像已被证明是一个有效的工具与附近的显微解剖分辨率的干细胞在体内的描述。为了检测与议员的干细胞，细胞与细胞特异性MR造影剂标记。为此，氧化铁纳米粒子，如超顺磁性氧化铁粒子（SPIO），适用的，因为他们的细胞检测，其优良的生物相容性的高灵敏度。超顺磁性氧化铁粒子组成的氧化铁核心和葡聚糖，carboxydextran或淀粉外衣，通过创建本地的外地的不均匀性，导致减少对T2加权MR图像信号的功能。本演示将展示技术与临床应用磁共振造影剂的干细胞，随后与磁共振成像的标记细胞在体内跟踪的非侵入性标签。

Watch this Scientific Journal Video about Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging at JoVE.com

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

对于新的干细胞疗法的评价，它是重要的非侵入性的追踪体内注射细胞。该视频将告诉你如何标注随后在体内磁共振成像体内的氧化铁造影剂的人类间质细胞和胚胎干细胞。

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 ...

labeling-hescs-hmscs-with-iron-oxide-nanoparticles-for-non-invasive

Research

JoVE Journal

Biology

9.8K 次观看.  Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco. 对于新的干细胞疗法的评价，它是重要的非侵入性的追踪体内注射细胞。该视频将告诉你如何标注随后在体内磁共振成像体内的氧化铁造影剂的人类间质细胞和胚胎干细胞。

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

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 ...

科研

生物学

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...