このプロジェクトは、再生医療のためのカリフォルニア工科大学からレオンJ.タールのSEEDの助成金によってサポートされていました。トビアスヘニングは、ドイツ研究協会（DFG、HE 4578/1-2）から奨学金研究によって資金を供給された。我々は感謝してヒト胚性幹細胞の培養上彼のアドバイスをファニートのMenesesを確認応答したい。

FerucarbotranとhMSCのラベリング これはFerucarbotran、MRイメージングのための酸化鉄ベースの造影剤でヒト間葉系幹細胞を標識するためのテクニックです。 <ol><li>開始するには、プレート18〜24時間、80％の合流点でT75フラスコ中のラベリング手順の前に細胞。別の培養皿を使用する場合、これは1cm 2当たり約10000細胞に等しくなります。 </li><li>翌日、無血清培地8mlをするResovistの30μlを添加してラベリングメディアの準備から始まります。これは100μgのFe / mlの培地の濃度に対応する、80％コンフルエントに一つT75フラスコにラベルを付けます。 </li><li>培地を外し、PBSまたは無血清培地で細胞を1回洗浄する。これは、残留血清タンパク質と造影剤と影響力の標識効率を結合することができるメディアの他の成分を取り除くために行われます。 </li><li>フラスコにラベルメディアを追加し、インキュベーターにフラスコを戻す。 </li><li> 2時間後、20％FCSの最終濃度が達成されるように、FCSの2ミリリットルを追加します。これは、細胞が分化して刺激を受けていないことを保証するために行われますが、代わりに使い慣れた環境で成長しています。 </li><li> 18時間にわたって細胞をインキュベートする。これは最高の一晩に行われます。 </li><li>翌日、PBSで細胞を洗い流した後、それらをいつものようにトリプシン処理。 </li><li>一度トリプシン、細胞懸濁液は、メディア内の残留、フリー造影剤を取り除くために3回洗浄する必要があります。これは、PBSで5分間で400 rcfで、細胞を遠心分離することで行われます。 </li><li>最終的に細胞ペレットを再懸濁し、さらなる実験のために準備ができます。 </li><li>一部は前の洗浄ステップの間に失われた可能性があるとして、現在のセルを数える。また、トリパンブルー排除アッセイを使用して、この時点で実行可能性のテストを実行し、標識効率を測定する分光分析のためにいくつかのサンプルを取る。 </li></ol> Ferumoxidesでヒト胚性幹細胞の標識 <ol><li>開始するには、プレート10cmの皿でヒトES細胞、いつものように。これらの料理は、ゼラチンをプレコートされ、それらの上に照射したフィーダー細胞を持っている。あなたにもフィーダーフリーの培養のためにこのプロトコルを使用することができます。 </li><li>彼らは中規模のコロニーを形成するようにヒトES細胞をアタッチし、約3-4日間に成長しましょう​​。この時間の間に、コロニーが大きいコロニーは後で分割することがより困難であるとして、大規模に成長していないことを確認してください。 </li><li>分化した細胞は、そのコロニーの清浄度に応じて、これらの培養物を汚染することができます、あなたは、解剖によって差別化された文化を取り除くために必要があります。 </li><li> 100μg/mlと完全なhESCのメディアの濃度でFerumoxidesから成るラベル、メディアを準備します。このため、我々は、89μlFerumoxidesと完全増殖培地10mlを混ぜる。 </li><li>完全なメディアで一度皿を洗ってください。 </li><li>皿あたりのメディアのラベル付けの10 mlを添加し、4時間細胞をインキュベートする。 </li><li>今、ラベルは完了です。次のステップでは、細胞を洗浄し、さらに使用するために、単一の細胞懸濁液を得ることが必要です。この場合、最初のPBSで皿を洗う。 </li><li>その後、0.25％トリプシン5mlのでPBSを交換し、5分間インキュベーター内でインキュベートする、または細胞がデタッチされるまで。それは頻繁に料理をタップするのに役立ちます。 </li><li>セルは切り離されている。料理は大きなコロニーが含まれていると残って塊があるかもしれないことに留意してください。 </li><li>これらの塊を取り除くために、いずれかの血清学的ピペットを使用して一度全部停止を混在させることができる、それは別の2分間放置します。 </li><li>我々は、細胞膜を破ることができる細いチップを通して、細胞のように繰り返し吸引をエッペンドルフピペットを使用しないでください。 </li><li>残留塊が存在する場合、一つは40μmのセルストレーナーを通して細胞懸濁液を実行することができます。 </li><li>すべてが正しく機能する場合、単一の細胞懸濁液はゼラチンコーティング、ESC -行列と死細胞に由来する、破片の多くと混合されている存在。このを取り除くために、5分間、400 rcfで、それらをスピンダウンして細胞を2回洗浄し、完全なメディアで再懸濁します。 </li><li>今、ESCは照射フィーダー細胞から分離する必要があります。これは、ゼラチンコートしたディッシュに細胞懸濁液をめっきすることによって効率的に行うことができます。 </li><li>料理が操作されていない場合は、すべての細胞が堆積物下になりますが、フィーダーはESCより速くアタッチされます。 </li><li>フィーダは主に料理に添付する必要がある一方そう、上清を45分後に撮影されている場合、それは、主にESCが含まれている必要があります。 </li><li>この方法では、さらなる実験のために使用することができる磁気標識されたヒト胚性幹細胞の単一細胞の懸濁液が得られる。 </li><li>以前のプロトコルと同様に、この時点では、セルをカウントし、ラベリング効率の実現可能性評価と測定用サンプルを採取する必要があります。 </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>

幹細胞の非侵襲的な描写は、ほぼすべての幹細胞ベースの治療を監視するために重要です。標識された幹細胞のin vivo動態ののより良い理解は、のようなMR画像上で可視化、合理的に方法および幹細胞ベースの治療をより効果的に使用する可能性があります。我々は臨床的に適用される造影剤とイメージング技術を使用しているため、私たちの提示プロトコルは、患者のアプリケーションに簡単に翻訳する必?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
<tr>
<td>FBS characterized</td>
<td>Reagent</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Cell Strainer (40 &#x03BC;m Nylon)</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>Knockout DMEM</td>
<td>Reagent</td>
<td>GIBCO</td>
<td>10829018</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>Glutamine</td>
<td>Reagent</td>
<td>GIBCO</td>
<td>25030081</td>
<td>&nbsp;</td>
<tr>
<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>
		</table>
		</div>

labeling hescs and hmscs with iron oxide nanoparticles for non-invasive in vivo tracking with mr imaging

近年では、幹細胞研究、発生生物学、様々な疾患と再生医療への影響の可能性についてのより良い理解につながっている。生体内で何度も移植された幹細胞を監視する非侵襲的方法は非常に我々の能力のメカニズムを制御する幹細胞の死を理解し、幹細胞の生着を​​向上させる栄養因子とシグナル伝達経路を特定するためにを高めるであろう。 MR画像は、近くに微細な解剖学的分解能を持つ幹細胞のin vivo描写のための効果的なツールであることが証明されています。 MRで幹細胞を検出するためには、細胞が細胞特異的MRの造影剤で標識する必要があります。この目的のために、このような超常磁性酸化鉄粒子（SPIO）のような酸化鉄ナノ粒子は、、のための細胞の検出とその優れた生体適合性のための彼らの高感度で、適用されます。 SPIO粒子は酸化鉄のコアとT2強調MR画像上で減少信号を発生するローカルフィールドの不均一性を、作成することによって、デキストラン、carboxydextranまたは澱粉コート、および関数で構成されています。このプレゼンテーションでは、MRイメージングと標識された細胞の生体内追跡、その後の非侵襲的に臨床的に適用可能なMR造影剤と幹細胞の標識のためのテクニックのデモンストレーションを行います。

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

新しい幹細胞療法の評価のためにそれはin vivoで注入された細胞を非侵襲的に追跡することが重要です。このビデオでは、生体内でのその後のMRイメージングのための生体内で酸化鉄ベースの造影剤によるヒト間葉や胚性幹細胞を標識する方法を紹介します。

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

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

9.7K ビュー.  Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco. 新しい幹細胞療法の評価のためにそれはin vivoで注入された細胞を非侵襲的に追跡することが重要です。このビデオでは、生体内でのその後のMRイメージングのための生体内で酸化鉄ベースの造影剤によるヒト間葉や胚性幹細胞を標識する方法を紹介します。

ビデオ: 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.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

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 ...

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.

シンクロトロンX線トモグラフィを用いたサブミクロン分解能を持つ非侵襲的3次元可視化

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 ...

Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12, behavioral approaches 13, 14,15, 16-18 and simulation 19, the use of mock scanner areas 20,21, basic relaxation 22 and a combination of these techniques 23 have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.

Making MR Imaging Child&#039;s Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure

Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children.

小児神経イメージングプロトコル、ガイドラインと手順 - MRイメージングチャイルドプレイを作る

Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural ...

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.

で小動物の筋肉の生着のダイナミクスの追跡インビボでの蛍光イメージング

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.

レーザーUncagableフルオレセインデキストランでゼブラフィッシュ細胞の系統ラベリング

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.

代謝標識とフローサイトメトリーで細胞周期の進行速度を測定

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.

ロイシンリッチの代謝標識は、放射性リン酸キナーゼ1および2を繰り返します

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.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

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.

ワンナノメートルの精度で蛍光イメージング（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.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

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