이 프로젝트는 재생 의료에 대한 캘리포니아 연구소에서 레온 J. Thal 시드 부여에 의해 지원되었다. 토바 이어스 헤닝는 독일 연구 협회 (DFG, HE 4578/1-2)에서 급비 연구에 의해 투자되었다. 우리는 기꺼이 그 인간의 배아 줄기 세포의 문화에 대한 그의 조언 후아의 Meneses을 인정 싶어요.

Ferucarbotran과 hMSC의 라벨링 이것은 Ferucarbotran, MR 영상을위한 철 산화물 기반의 콘트라스트 에이전트 인간 mesenchymal 줄기 세포를 라벨에 대한 기술입니다 <ol><li> 시작하려면, 플레이트 18~24시간 80 % 합류에 T75 flasks의 라벨 절차 이전에 세포. 다른 문화 접시를 사용하는 경우이 cm 2 당 10000에 대한 세포 같습니다. </li><li> 그 다음날, 혈청 무료 미디어 8 ML에 Resovist 30 μl를 추가하여 라벨 미디어를 준비로 시작합니다. 이것은 100 μg 철 / ML 미디어의 농도에 해당, confluency 80% 하나 T75 플라스크 레이블을 것입니다. </li><li> 문화 미디어를 벗고 PBS 또는 혈청 무료 미디어 번 세포를 씻으십시오. 이것은 잔류 혈청 단백질과 대비 에이전트와 영향력 라벨 효율을 바인딩할 수있는 미디어의 다른 성분을 제거하기위한 것입니다. </li><li> 술병에 상표 미디어를 추가하고 인큐베이터에있는 술병을 돌려 넣어. </li><li> 2 시간 후에, 20 % FCS의 최종 농도가 달성되도록 FCS의 2ml를 추가합니다. 이것은 세포가 차별 자극을받지 않도록 할, 대신 자신의 친숙한 환경에서 성장합니다. </li><li> 18시간을 통해 세포를 품어. 이것은 최고의 하룻밤 완료됩니다. </li><li> 다음날, PBS로 세포를 씻어 후, 그들이 평소 trypsinize. </li><li> 일단 trypsinized, 세포 현탁액은 미디어 잔류, 무료 대비 에이전트 없애 3 번 세탁해야합니다. 이것은 PBS 5 분 400 RCF에서 세포를 centrifuging하여 이루어집니다. </li><li> 최종 세포 펠렛은 resuspended 더욱 실험을 준비하실 수 있습니다. </li><li> 일부는 이전의 세척 단계 중 손실되었을 수도로서 세포를 계산합니다. 또한, Trypan 파랑 제외 분석을 사용하는이 시점에서 생존 테스트를 수행하고 라벨 효율을 측정하는 spectrometric 분석을위한 샘플을 채취. </li></ol> Ferumoxides와 인간 배아 줄기 세포의 라벨링 <ol><li> 시작하려면, 플레이트 10cm 요리에 hESC, 평소처럼. 이들 요리는 젤라틴으로 미리 코팅하고 그들에게 조사 피더 세포를합니다. 당신은뿐만 아니라 피더없는 문화에 대해이 프로토콜을 사용할 수 있습니다. </li><li> 그들은 중간 크기의 식민지를 형성 있도록 hESC는 첨부하여 약 3-4일에 대한 성장하자. 큰 식민지 나중에 이혼하는 것이 더 어려울 있으므로이 기간 동안, 식민지 큰 성장을하지 않도록. </li><li> 차별화된 세포 너무 콜로니의 청결에 따라, 이러한 문화를 오염시킬 수있다, 당신은 절개로 차별 문화를 없애해야 할 수도 있습니다. </li><li> 100μg/ml 및 전체 hESC 미디어의 농도에 Ferumoxides 구성되어 라벨 미디어를 준비합니다. 이를 위해, 우리는 89μl Ferumoxides 및 전체 성장 미디어 10 ML을 섞는다. </li><li> 전체 미디어와 한 접시를 씻으십시오. </li><li> 요리마다 미디어를 라벨링 10 ML을 추가하고 4 시간 동안 세포를 품어. </li><li> 이제 라벨이 완료됩니다. 다음 단계에서, 그것은 세포를 씻어 더욱 사용하기 위해 단일 ​​세포 현탁액을하는 것이 필요합니다. 이를 위해 먼저 PBS로 접시를 씻어. </li><li> 그런 다음, 0.25 %의 트립신의 5ml으로 PBS를 교체 5 분 동안 인큐베이터에서 부화하거나, 세포가 분리까지. 그것은 자주 접시를 도청하는 데 도움이됩니다. </li><li> 세포는 이제 분리했습니다. 요리가 큰 식민지가 포함된 경우 남은 대단히 짧은 시간이있을 것을 명심하십시오. </li><li> 이 대단히 짧은 시간을 없애, 하나 serological 피펫을 사용하여 한 번 전체 중지를 섞을 수 있습니다 다음, 그것이 다른 2 분 앉아 보자. </li><li> 우리는 세포 세포막을 깰 수있는 얇은 팁을 통해 세포의 반복으로 빨아을 Eppendorf 피펫을 사용하지 마십시오. </li><li> 잔여 대단히 짧은 시간이 존재할 경우, 하나는 40μm 셀 스트레이너를 통해 세포 현탁액을 실행할 수 있습니다. </li><li> 모든 올바르게 작동하는 경우, 단일 세포 현탁액 이제 ESC 매트릭스와 죽은 세포 젤라틴 코팅에서 발생하는, 파편의 많은 혼합되어 존재한다. 이 없애 5 분 400 RCF에서 그들을 회전하여 두 세포를 씻어 및 전체 미디어에서 그들을 resuspend. </li><li> 자, ESC는 조사 피더 세포로부터 격리해야합니다. 이것은 젤라틴 - 코팅 접시에 세포 현탁액을 도금하여 효율적으로 수행할 수 있습니다. </li><li> 접시가 조작하지 않으면, 모든 전지는 퇴적물 아래되지만 피더는 ESC보다 빠르게 연결할 수 있습니다. </li><li> 모이통가 주로 요리에 연결되어 있어야하는 동안, 그래서 뜨는 45 분 후에 이륙있다면, 그것은 주로 ESC를 포함해야합니다. </li><li> 이 방법은 추가 실험을 위해 사용할 수있는 자기 (磁 气)로 분류 인간의 배아 줄기 세포의 단일 세포 현탁액이 얻어진다. </li><li> 이전 프로토콜과 마찬가지로,이 시점에서, 당신은 세포를 계산하고 라벨 효율성 생존 능력의 평가와 측정을위한 샘플을 채취해야합니다. </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>

줄기 세포의 비침습 묘사는 거의 모든 줄기 세포 기반 치료를 모니터링 중요합니다. 표시 줄기 세포의 생체내의 속도론에의 더 나은 이해로 MR 이미지에 대한 시각, 합리적인 방법과 줄기 세포 기반 요법보다 효과적인 사용을 초래할 수 있습니다. 우리가 임상 적용 대조 대리인 및 이미징 기술을 사용 이후, 우리의 제공 프로토콜은 환자의 어플 리케이션에 쉽게 번역되어야합니다. 우리의 방법의 잠재적인 임상 ?...

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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

최근에는 줄기 세포 연구는 발달 생물학, 각종 질병 및 재생 의료에 미치는 잠재적인 영향을 더 잘 이해하게되었다. 생체내에서 반복 이식 줄기 세포를 모니터링하는 비침습 방법은 크게 메커니즘을 이해하는 능력을 향상시킬 것이라는 제어 줄기 세포 죽음과 줄기 세포 engraftment을 향상 영양 요인과 신호 경로를 확인합니다. MR 영상은 근처의 미세​​한 해부 해상도 줄기 세포의 생체내 묘사에 대한 효과적인 도구로 입증되었습니다. MR과 함께 줄기 세포를 감지하기 위해서는 세포가 세포 특정 MR 대비 요원으로 표시해야합니다. 이 목적 들어, superparamagnetic의 철 산화물 입자 (SPIO)으로 철 산화물 nanoparticles은, 때문에 세포 검출과 우수한 biocompatibility 위해 고감도의 적용됩니다. SPIO 입자는 철 산화물 코어와 T2 - 가중 MR 영상에서 감소 신호를 발생 지역 현장 inhomogeneities를 만들어 dextran, carboxydextran 또는 전분 코팅, 그리고 기능으로 구성되어 있습니다. 이 프레 젠 테이션은 MR 영상과 레이블 세포의 생체내 추적에 후속 비침습에 대한 임상 적용 MR 대비 에이전트와 함께 줄기 세포의 라벨에 대한 기술을 설명합니다.

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

새로운 줄기 세포 요법의 평가는 생체내에 주입된 세포를 추적 비 invasively하는 것이 중요합니다. 이 동영상은 생체내의 후속 MR 영상에 대한 생체내의 철 산화물 기반의 콘트라스트 에이전트와 인간 mesenchymal와 배아 줄기 세포를 레이블하는 방법을 보여줍니다.

이런 산화물로 hESCs과 hMSCs을 라벨링하는 MR 영상과 생체내 추적 비침습에 대한 Nanoparticles

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 조회수.  Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco. 새로운 줄기 세포 요법의 평가는 생체내에 주입된 세포를 추적 비 invasively하는 것이 중요합니다. 이 동영상은 생체내의 후속 MR 영상에 대한 생체내의 철 산화물 기반의 콘트라스트 에이전트와 인간 mesenchymal와 배아 줄기 세포를 레이블하는 방법을 보여줍니다.

비디오: 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.

광학 이미징과 비침습 감지를위한 형광 염료와 함께 줄기 세포를 라벨링

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 ...

연구

생물학

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.

싱크로 트론 - X - 레이 - tomography를 사용하여 서브 마이크론 해상도로 비침습 3D - 시각화

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.

소아 Neuroimaging 프로토콜, 지침 및 절차 - MR 이미징 아동의 놀이 만들기

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.

작은 동물의 근육 Engraftment의 역학에 의해 추적 VIVO에서 형광 이미징

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.

레이저 Uncagable 플루오레신 Dextran과 Zebrafish 세포의 리니지 라벨

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.

신진 대사 레이블 및 유동세포계측법와 세포주기 진행의 동력학 측정

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.

류신 리치의 신진 대사 라벨링 방사성 인산과 키니 아제 1과 2를 반복

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.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

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.

하나 나노 미터의 정확도와 형광 이미징 (피오나)

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.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

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