我们想感谢在笼中的荧光素丹卡林帮助。此外，我们想感谢以下资金来源：范德比尔特大学医学发育生物学培训资助计划学校，江淮，和美国国立卫生研究院HD054534to JTG。

1。笼中的荧光素葡聚糖的合成<ol><li>测量出3.5 - aminodextran 4毫克，并添加到含1毫克的CMNB笼荧光SE的Invitrogen公司提供的有色管。在我们的手中，这一比例给出了一个〜2.5％，染料分子右旋糖酐的平均装载。 </li><li>加入500μL0.1 M的NA 2 B 4 O 7（四硼酸钠）缓冲区管。 </li><li>第旋涡30秒解散aminodextran笼荧光。 </li><li>让我们在涡旋混合器反应过夜。 </li><li>拧断则巴自旋列底部封闭和放松的上限。标记一个点上用毡尖笔列。将列在15 mL锥形管。 </li><li>在1000 XG离心机面临的转子中心的点为2分钟。压实后，立即使用列。 </li><li>放置在一个新的15 mL锥形管压实列。 </li><li>池反应混合物压实列树脂床的中心转移到。更换盖。 </li><li>在1000 XG离心机面临的转子中心的点为2分钟。分拆后，洗脱液和列的顶部将大致颜色相同的黄色阴影。 </li><li>黄色洗脱液（〜350μL）转移到1.5 ml离心管。 </li><li>冻干以干燥在speedvac。盖用铝箔纸speedvac。 </li><li>重新暂停导致〜苍白黄泡在水中2-3毫克到终浓度为1％W / V </li><li>在铝箔盖管，储存在-20 ° C的这一解决方案。这是一个好主意，以等分葡聚糖的解决方案，以防止重复冻融。 </li></ol> 2。笼中的荧光素葡聚糖注射到斑马鱼胚胎<ol><li>在0.2M氯化钾稀释1％W / V股票笼荧光葡聚糖1:5或1:10。 </li><li>注入0.5 - 笼入蛋黄细胞阶段的胚胎使用高压注射器的荧光解决方案1.0nL。一旦注入，胚胎离合器必须保持在28.5 ° C至减少非特异性uncaging在黑暗中。 </li><li>手动移除胚胎chorions。 </li><li>麻醉在4％tricaine的胚胎。 </li><li>胚胎将被安装在0.8％琼脂糖35mmx10mm培养皿中，然后盖上蛋水。 </li><li>至少15分钟，在50 ° C的热块平衡融化的琼脂糖。 </li><li>使用的玻璃吸管，吸取少量的水的一个胚胎，并释放到琼脂糖注：这是很重要的冷却琼脂糖管你的手，约30秒前把该管的胚胎，以避免杀死胚胎。 </li><li>移液器从试管胚胎，约琼脂糖50μL和弹出到培养皿的中心内容。 </li><li>快速定位与uncaged面临细胞的胚胎，使用一个塑料螺纹注 ：酷很快，所以仓促定向胚胎琼脂糖。如果操纵琼脂糖变得僵硬后，可能会损坏胚胎。 </li><li>允许琼脂糖完全巩固（这需要几分钟），并填写菜三分之二与鸡蛋的水含有tricaine 4％和0.003％机动部队。 </li></ol> 3。激光荧​​光素葡聚糖Uncaging <ol><li> uncaging使用显微镜必须有一个40倍的水浸泡的目标。 </li><li>我们用365 nm的染料细胞和70％/ 30％的分裂分色镜的光电子仪器的激光。 </li><li> uncaging之前，激光必须作出的目标和衰减到合适的电源的齐焦。 </li><li>要集中的激光，将根据客观镜像玻片上，重点目标，直到在幻灯片的划痕可见。 </li><li>调整激光衰减器，直到激光可以产生清晰可见，在单脉冲镜从头。 </li><li>向上和向下调整的目标重点。如果激光可以产生了一个从无到有的目标是重点，调整激光的聚焦¼交替上升或下降。重复，直到激光可以从头开始的目标是只有当聚焦镜。 </li><li>根据uncaged面积的目标和重点地点安装胚胎。 </li><li>要uncage，着眼于利益的一个单元格，并在2-3赫兹脉冲的10-20倍。 </li><li> uncaging后，免费的胚胎剥离琼脂糖钳和把鸡蛋水含有0.003％机动部队。让胚胎的发展，直到它们被固定在光紧框。 </li></ol> 4。抗体标记的Uncaged荧光素葡聚糖检测<ol><li>修复胚胎在AB固定液（4％多聚甲醛，氯化钙为0.3mm 2，8％的蔗糖，1X的磷酸盐缓冲生理盐水，pH值7.3），两到四个小时，在室温（RT）或隔夜（O / N）在4 ° C所有标签的​​步骤，应保持轻紧框或铝箔包裹管的胚胎。 </li><li>删除固定液，用100％甲醇取代，在室温下10分钟离开，除去甲醇和replaCE新鲜100％甲醇。 </li><li>商店在-20 ° C，在甲醇至少30 分钟注 ：您可以存储长达两个星期之前抗原表位在甲醇开始降低。 </li><li>补充水分胚胎在室温洗涤5分钟，75％MeOH/1X磷酸盐缓冲生理盐水，0.1％吐温20（1X PBSTw），然后50％MeOH/50％1XPBTw，然后MeOH/75％1X PBSTw 25％。 </li><li>在1XPBSTw 5分钟内清洗三次。 </li><li>如果胚胎&gt; 24小时，permiabilize 10μg/ mL的蛋白酶K在1XPBSTw胚胎。 Permiabilization时间是在舞台上的依赖。五分钟蛋白酶K是足够的，如果胚胎受精后（HPF）是24至48小时。如果年纪较大的幼虫，可能需要更多的时间。重新修复抗体固定液在室温下20分钟</li><li>在1XPBSTw 5分钟，在室温下清洗5次。 </li><li>孵育块解决方案（1XPBS，0.1％的Triton X100，10％羊血清，10％的牛血清蛋白，1％二甲基亚砜）在室温下1-2小时。 </li><li>封闭液稀释的抗体（抗荧光素加抗细胞类型特异性标志物抗体）。 </li><li>孵育胚胎O / N在4 ° C在含初级抗体的解决方案。 </li><li>清洗四次，在1XPhosphate 20分钟的缓冲生理盐水，0.1％的Triton X100（PBSTx）在室温下。 </li><li>在封闭液稀释的荧光标记抗体（通常为1:300）。 </li><li>孵育O / N在4 ° C在含有二级抗体的解决方案。 </li><li>在室温下在1XPBSTx 20分钟清洗四次。 </li><li>清除在50％glycerol/50％，在室温下至少有两个小时1XPBS的胚胎，然后取出甘油/ PBS混合，添加100％的甘油，让站在O / N 4 ° C。 </li></ol> 5。代表性的成果： 免疫标记后，应该有一个uncaged（Figure.1）细胞染色丰富的区域。这是很平常看到一个比较高的信噪比&gt; 48小时老斑马鱼，由于荧光素的自发uncaging。 <img src="/files/ftp_upload/2672/2672fig1.jpg" alt="图1" /> 图1。传承的斑马鱼松果体复杂的标签。 HPF，24岁的前松果体原基的部分，foxd3表示：GFP的表达，uncaged使用紫外激光脉冲通过48hpf），左路集群的细胞称为parapineal（红色圆圈）已经摆脱了uncaged域（白色圆圈），B）Uncaged的荧光信号是丰富parapineal（红圈）和松果体器官（白色圆圈） 。

<ol>
	<li>Hatada, Y., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fate+map+of+the+epiblast+of+the+early+chick+embryo.">A fate map of the epiblast of the early chick embryo.</a> Development. 120, 2879-2889 (1994).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell-stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell-stage Xenopus embryo.</a> Dev Biol. 122, 300-319 (1987).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+Precursors+Can+Give+Rise+to+All+Major+Cell-Types+of+the+Frog+Retina.">Multipotent Precursors Can Give Rise to All Major Cell-Types of the Frog Retina.</a> Science. 239, 1142-1145 (1988).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+fluorescent+tracers+to+study+neural+cell+lineages.">Microinjection of fluorescent tracers to study neural cell lineages.</a> Development. , 1-8 (1991).</li><li>Woo, K., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system.">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc Natl Acad Sci U S A. 99, 12651-12656 (2002).</li><li>Sato, T., Takahoko, M., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuC:Kaede,+a+useful+tool+to+label+neural+morphologies+in+networks+in+vivo.">HuC:Kaede, a useful tool to label neural morphologies in networks in vivo.</a> Genesis. 44, 136-142 (2006).</li><li>Wiedenmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc Natl Acad Sci U S A. 101, 15905-15910 (2004).</li><li>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Fluorescent Probes and Research Chemicals. , 707-711 (2002).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75, 551-562 (1997).</li><li>Kozlowski, D. J., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+(caged)+fluorescein+as+a+cell+tracer+for+fate+mapping+in+the+zebrafish+embryo.">Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo.</a> Methods Mol Biol. 135, 349-355 (2000).</li><li>Russek-Blum, N., Nabel-Rosen, H., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fate+map+of+the+zebrafish+diencephalon.">High resolution fate map of the zebrafish diencephalon.</a> Dev Dyn. 238, 1827-1835 (2009).</li><li>Summers, R. G., Piston, D. W., Harris, K. M., Morrill, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orientation+of+first+cleavage+in+the+sea+urchin+embryo,+Lytechinus+variegatus,+does+not+specify+the+axes+of+bilateral+symmetry.">The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.</a> Dev Biol. 175, 177-183 (1996).</li></ol>

如上所述，这个协议提供了相对快速的血统在斑马鱼标记法，显微注射法，显微镜，免疫后的常用技巧。我们发现，激光uncaging是最有效和最具成本效益的方式uncage在本地化时尚的荧光。这种方法可用于实验端点4 DPF后期谱系标签。然而，细胞分裂时，每个单元的笼荧光素浓度，最终跌幅超过检测水平。此外，已经出现了自发的，非特异性荧光uncaging的斑马鱼幼虫 11的报告。因此，u​​ncag...

A correction was made to <a href="http://www.jove.com/details.php?id=2672">Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran</a>. There was an error in the authors, Ilya A. Shestopalov and James K. Chen, names. The author's names have been corrected to: Ilya A. Shestopalov and James K. Chen instead of: Ilya Shestopalov and James Chen

A correction to the article Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran was made.

Erratum: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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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>CMNB-caged fluorescein SE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C20050</td>
<td>&nbsp;</td>
<tr>
<td>Aminodextran, 10kDa</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D1860</td>
<td>&nbsp;</td>
<tr>
<td>Zeba desalt spin columns</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>89889</td>
<td>&nbsp;</td>
<tr>
<td>goat anti-fluorescein antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A11095</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 633 Donkey anti-goat antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A21082</td>
<td>&nbsp;</td>
<tr>
<td>35mmx10mm petri dishes</td>
<td>&nbsp;</td>
<td>Becton-Dickinson</td>
<td>351008</td>
<td>&nbsp;</td>
<tr>
<td>Upright compound microscope with 40x water immersion objective</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DM6000B</td>
<td>&nbsp;</td>
<tr>
<td>MicroPoint Manual Laser Illumination System</td>
<td>&nbsp;</td>
<td>Photonic Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Alfa Aesar</td>
<td>L06690</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E10521</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115836991</td>
<td>&nbsp;</td>
<tr>
<td>Plastic thread</td>
<td>&nbsp;</td>
<td>Any sewing supply store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Agarose</td>
<td>&nbsp;</td>
<td>Research Products International</td>
<td>A20090</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>Savant SPD131DDA</td>
<td>&nbsp;</td>
<tr>
<td>Vortexing mixer</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>Vortex Genie 2</td>
<td>&nbsp;</td>
<tr>
<td>Pressure injector</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>MPPI-3</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

lineage labeling of zebrafish cells with laser uncagable fluorescein dextran

发育生物学中的一个核心问题是演绎出无数的细胞类型，目前他们未分化的前体出现在脊椎动物的起源。研究人员所采用的谱系标签的各种方法，如DII 标签 1和压力注射溯源酶 2的模型系统开发的后期阶段，以确定细胞的命运。在斑马鱼（ 斑马鱼 ）的第一命运地图iontophoretic注射荧光染料如罗丹明右旋糖酐，组装，成单细胞在胚胎的离散地区和追踪标记细胞的命运，随着时间的推移3-5。这些方法虽然有效，技术要求高，需要专门的设备，不常用的斑马鱼实验室发现。最近，photoconvertable荧光蛋白，如EOS和枫，不可逆转的交换机从绿色变为红色荧光，当暴露在紫外线，增加使用在斑马鱼 6-8 。光学清晰度的转基因斑马鱼的胚胎和相对缓和这些谱系标签特别有吸引力的工具，并观察细胞的迁移，在体内7。尽管它们的效用，这些蛋白质有一些缺点相比，染料介导的宗族标记方法。最关键的是我们在发现这些蛋白质的光转化过程中获得3 - D分辨率高的困难。在这一点上，也许在斑马鱼的血统标签的决议和易用性的最佳结合 ​​，使得使用笼荧光葡聚糖，一种荧光染料，势必淬火组口罩荧光9。染料就可以“uncaged”（从淬火组发布）在一个特定的细胞使用激光或水银灯紫外线光，使荧光或免疫检测的可视化。笼荧光iontophoretic方法不同，可注射用标准的注射器具，并配备了一个针孔10 epifluorescence显微镜uncaged。此外，对荧光抗体检测只有uncaged形式，和抗原表位生存固定以及11。最后，笼中的荧光可以被激活，具有非常高的3 - D分辨率，特别是如果采用双光子显微镜12,13。这个协议描述笼荧光和激光uncaging宗族标签的方法。 Subsequenctly，uncaged荧光检测如GFP的其他抗原表位，同时通过与抗体标签。

Watch this Scientific Journal Video about Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran at JoVE.com

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

该协议描绘的方式，标签和跟踪的小斑马鱼的胚胎细胞群体，使用紫外线uncaging笼荧光，整个装载免疫标记uncaged荧光信号放大的命运。

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 ...

lineage-labeling-zebrafish-cells-with-laser-uncagable-fluorescein

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13.8K 次观看.  Vanderbilt University. 该协议描绘的方式，标签和跟踪的小斑马鱼的胚胎细胞群体，使用紫外线uncaging笼荧光，整个装载免疫标记uncaged荧光信号放大的命运。

视频: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

脂筏的测定分区在活细胞中的荧光标记探针的荧光相关光谱（FCS）

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 &#181;m is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.
To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.
The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

An original experimental setup for heating cells in a culture dish using 1.94 &#181;m continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

连续波ium激光加热培养细胞以研究细胞热效应

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...