成像进行了一个堂堂正正的徕卡SP5共聚焦显微镜位于伦敦帝国学院的电影工厂，由马丁·Spitaler博士管理的范围内。 试样夹持器和头盔是在与英国伦敦帝国学院的化学工程系合作，与意见塞缪尔·琼斯和西蒙·舒尔茨博士。 尼古拉Ruivo，弗朗西斯科·迪亚兹和哥伦比亚广播公司的工作人员在国子监是有助他们对小鼠饲养援助和咨询。 马克·斯科特资助HFSP和电影，Olufolake Akinduro由CRUK和克里斯蒂娜罗塞尔索由CRUK，KKLF，BBSRC和HFSP。

所有涉及使用动物的程序，是根据当地法规进行。我们的工作是经英国内政部以及国子监伦理审查小组。允许程序项目的许可文档中描述，并按照指引，以确保动物在任何时候的福利。确保遵守有关地方进行工作的国家实验动物的立法。 1，标签和注塑HSPCs的： <ol><li>所描述的罗塞尔索14-15收获细胞。 </li><li>制备的细胞以10 6个细胞/ PBS中ml的浓度。注意：使用血细胞计数器或由细胞分选器所提供的信息来计算的单元格;被标记，并注射细胞的数目而变化取决于细胞类型（污点例如10,000 - 20000的HSCs，100000 - 300000的HPC）;如果少于10 5个细胞，重悬在100μlPBS中的工作;具有细胞在PBS中没有任何血清是很重要的，因为血清抑制染色。 </li><li>加确实在细胞悬浮液以5μM的最终浓度，并立即涡旋该悬浮液，以确保该染料不沉淀出的溶液和未标记的细胞。 </li><li>孵育10分钟，在37℃，然后通过在500 XG旋转5分钟，倾出液体，并重新悬浮于适当体积的沉淀物为静脉注射（〜100 - 150微升）洗</li><li>将细胞重悬于200μl的PBS中，并收集到胰岛素注射器。注：胰岛素注射器，推荐通过常规注射器，因为它没有针死空间，因此，可以管理整个细胞悬浮于鼠标。 </li><li>注入到细胞经尾静脉注射致死剂量照射小鼠。注：辐照已经公知，在骨髓微环境（包括造血细胞和基质成分），其发展比TI相当大的影响我。因此，它是非常重要的，以保持对辐射一致的定时，细胞注射（4至24小时，从照射最佳植入）和成像。在这里，照射进行注射前6小时，成像注射后进行20小时。 <ol><li>鼠标放置在温暖的室内（37℃），直到尾静脉出现vasodilated。 </li><li>将鼠标移动到限制器，小心地将针头插入尾静脉。 </li><li>注入细胞进入血管 - 注射无阻力应该会遇到。 </li><li>离开鼠标O 2 / N，以使细胞迁移到骨髓空格。 </li></ol></li></ol> 2，准备鼠标成像<ol><li>高压灭菌一双细镊子和一对精致的剪刀，以及在使用之前头盔和存储在一个无菌的环境。 </li><li>化妆麻醉尽可能新鲜，（0.38毫升氯胺酮+0.25毫升美托咪定+4.47毫升水）。注：OT她的批准麻醉药，包括异氟醚和其他可注射的，将是有效的了。描述的鸡尾酒是要在每10g体重0.1毫升腹膜内给药时，为30充值 - 加入50μl给药，每45分钟至1小时。 </li><li>交换机的双光子激光如有需要，然后开始在显微镜和软件，校准阶段的提示时。交换机上的激光器，让他们热身和准备，同时鼠标的成像稳定。 </li><li>麻醉用准备好的麻醉剂的混合物（步骤2.2），通过腹腔注射麻醉选择鼠标。现在定期在整个协议的监控，通过脚踏板反射的发生深麻醉。注意：鼠标必须小心整个过程进行监视，如果麻醉似乎减轻（45通常后 - 60分钟），然后施用顶式剂量（如2.2详细说明）。希望不同年龄，不同性别的老鼠之间的一些差异。这是祁门功夫，功夫坦讨论与您当地的兽医官麻醉协议，以确保有足够的预防措施，以确保鼠标的福利。 </li><li>拭带的组织的70％乙醇头皮的顶部，确保没有得到任何乙醇在小鼠的眼中。 </li><li>使用无菌镊子和剪刀，小心地取出头皮的中央部分，以暴露颅骨面积要成像：通过解除皮肤与钳子作一小切口，在两耳之间的头部的背面。同时保持皮肤时，滑动剪刀的皮肤下，并沿着期望的成像区域的外侧轻轻地切割。 </li><li>擦拭骨外露，用无菌棉签蘸无菌PBS，以保持湿润。 </li><li>首先将金属头片的鼠标准备用于成像的头骨。 <ol><li>混合牙科用粘固剂的足够量的称重舟，直到它变成糊状物，并迅速应用到将连接到S的头盔的底部表面库尔。 </li><li>在水泥台，将头盔上鼠标的头骨，确保不要让对成像区域中的任何牙科水泥，然后等待它来设置。 </li><li>取少量站点内水凝胶这使头骨湿润。 </li></ol></li><li>附加的头盔在支架和使用螺钉，确保槽的支架凹槽内适合固定到位。 </li><li>转移到显微镜前，取下颅骨，用无菌棉签和清洁用无菌PBS的水凝胶。 </li><li>将支架插入显微镜阶段，根据鼠标位置的加热垫，插入直肠体温计探头和安全的一切都发生在用胶带阶段。 </li><li>将一小滴眼药膏对鼠标的眼睛，以确保他们不会干出在麻醉状态下。注意：鼠标不能独留在任何时间在成像过程中，同时在麻醉状态下。 </li></ol>体内成像：高分辨率栈和定时采集<ol><li>注重通过眼睛片头骨。 <ol><li>用纯化的，无菌的水，并用水浸渍透镜填充成像窗口，降低它，使得它接触的水滴。 </li><li>专注于使用显微镜目镜头骨的顶部，使用外部灯作为光源。 </li><li>定位在成像区域上的中央缝合并朝着头部的后部，以找到冠状缝的起始位置。 </li></ol></li><li>设置在显微镜以允许在激发和发射表中指定的相关荧光的有效激发和检测。注意：当莱卡SP5共聚焦直立与传统振镜扫描头在运行LAS-AF的软件平台用在这里，程序可以很容易地在其他显微镜/软件平台复制。这里使用的镜头是徕卡HCX IRAPOŁ25X瓦/ 0.95 NA b都可以用合适的放大倍率和高NA其他厂商UT相当于镜头<ol><li>把软件变成一种模式来捕捉这两个XY图像以及三维Z堆栈，并设置成像速度为400赫兹，分辨率为512×512像素。注：安装和硬件在这里导致鉴于620微米的领域，这可能与在其他平台上。 </li><li>置的软件，以使多个通道/轨道都能够被捕获，以及确保该集合的方法被设置为改变堆栈如果可能或帧之间设置在最低限度。设置3个独立的捕获设置，在关闭激光照射在采集每个堆栈的末端。注：三个通道将被要求在此过程中，以减少通道间的串扰。 </li><li>设置为第一顺序扫描的设置双光子骨质倍频信号（840 nm激发; 400 - 440 nm发射）;打开快门用于MP激光和确保MP增益和偏移正确的设置，激光功率为12.5 - 25％，而激光接通。选择一个合适的光电倍增管作为唯一的检测，改变颜色为白色。注意：NDD检测器与相应的过滤器应当用于骨检测时可实现更明亮，更深入的信号。 </li><li>用绿色荧光蛋白通道组合重复的自体荧光通道设置过程（543 nm激发; 560 - 600nm的发射），做（633 nm激发; 650 - 720 nm发射）下第二个设置进行扫描，利用两个相应的光电倍增管。改变通道的颜色以绿色环保为自体荧光和红色的了。注意：在使用绿色作为自发荧光通道允许更容易的检测没阳性细胞时，两个通道重叠 - 自体荧光的细胞显示为黄色/橙色由于在两个通道同时又做了细胞仅显示为红色，因为它们只出现在做渠道。自发荧光通道仅用于该比较，并且不以任何最终分析中使用，但是，如果用户希望，这是可以改变的，以不同的颜色显示的目的，以避免与GFP通道混乱。 </li><li>最后，建立了绿色荧光蛋白的第三个和最后扫描（488 nm激发; 500 - 530 nm发射）;确保在快门开启（如果需要），然后将488 nm激光线的激光功率为15％左右，或使用适当水平的激光，选择一个合适的光电倍增管作为唯一主动检测并改变其伪绿色。 </li><li>激活一个&#39;活&#39;的成像模式，开始选择的频道的预览扫描和检测调整增益和失调的最佳曝光。重复此步骤，在连续的窗口每一次扫描。 </li><li>保存这些多通道扫描，便于重复使用的设置。注：本允许用户加载在以后的会话的设置。 </li></ol></li><li>激活多工位捕捉，称为“马克ND查找“中所表现出的软件，并重新设置坐标点。 </li><li>扫描骨显像区，中央和冠状缝之间的交集出发，同时使用自体荧光扫描骨髓区域的深度（伪彩色绿色），做（假色红色）与复合视图。注：自体荧光的细胞将存在于两个信道，并出现橙/黄色的合成图像，而没标记的细胞将显示为红色，只有细胞的合成图像。 </li><li>当检测到一个单元格，单击“添加新位置图标的左侧”马克和查找“窗口，标志着这是一个新的坐标位置（x，y和z）在”马克和查找“工具。 </li><li>当针对当前场进行过检查，动视或左/右/上/下一个场的舞台的距离。重复这个过程对于每个视场，围绕整个成像窗口面积逐渐努力个左ê中央缝合，迈向鼠标的鼻子。一旦中央缝合分叉是在视线，移动到右侧的缝线，并重复该过程扫描左边沿相反方向（ 即 ，朝着冠状缝）。标记感兴趣的任何新的细胞使用的坐标&#39;标志，并查找“工具。 </li><li>一旦扫描成像窗口完成后，使用“标志，并查找”工具来查看点（选择所需的点并单独查看每个点）。设置每个单元的每个位置绕Z堆栈的顶部和底部，（许多软件平台，可以让你在每个位置上，从而降低了捕获空进行独立的Z-堆栈）。对于每一个点上，集中上下集的3D Z堆栈捕捉顶部和底部的Z轴位置，并在更新个人点&#39;标志，并查找“。设置的Z-Stack的时间间隔为5微米，平均每个顺序扫描通道，适当的质量：O线ř帧的平均水平。注意：增加扫描次数进行平均增加了采集时间长度。 </li><li>获得每个点的通过启动整个扫描过程感兴趣的高质量参考栈（通常被称为“启动”，而不是“捕获”）。注意：此扫描可作为未来高速成像的参考。 </li><li>一旦完成了高质量的扫描，激活延时设置捕获。 </li><li>在时间推移的设置，设置的时间间隔为5分钟，总运行时间为所需的长度（ 例如 ，5小时）。 “应用”这些设置进行全盘扫描。注意：为了减少这种扫描时间，以允许5分钟的时间流逝的时间间隔，人们可能需要减少用于平均的扫描次数，限制了分辨率为512×512象素，扫描转换为双向（有需要相位校正对准两个扫描方向），和/或增大扫描速度，以600赫兹或以上（超过600赫兹将导致变焦的成像区域的用于证明软件平台）。同样重要的是要注意，当一个连续的成像模式是用于最初获得高质量的图像叠加，同时采集时所用的时间推移成像过程中，以增加速度 - 这会导致信道之间的一些轻微的串扰它额外地在准确地分离参考栈的初始捕获重要性。 </li><li>开始成像（如前通过单击演示软件“开始”按钮）。注：请务必通过在需要的时候再给予，较小剂量，注意不要过量鼠标来维持麻醉的适当水平，定期补足水分，确保目标不干裂，监测生命体征和加热垫温度贯穿始终。 </li><li>完成后，由持有人提出的镜头，然后从显微镜载物台的支架，以确保从直肠探头和小时线吃席不被损坏。 </li><li>从舞台支架旋出头盔，小心地取出鼠标。使用颈椎脱位剔除小鼠，并通过捕捉它从头骨取出金属头件。将数据保存到磁盘，然后传输到服务器以供日后分析。注：头一件是由浸在丙酮中了几个小时，最后擦残留的水泥掉理想的清洁。 </li></ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Relationship+Between+Bone,+Hemopoietic+Stem+Cells+and+Vasculature.">The Relationship Between Bone, Hemopoietic Stem Cells and Vasculature.</a> Blood. 118 (6), 1516-1524 (2011).</li><li>Nombela-Arrieta, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Imaging+of+Haematopoietic+Stem+and+Progenitor+Cell+Localization+and+Hypoxic+Status+in+the+Bone+Marrow+Microenvironment.">Quantitative Imaging of Haematopoietic Stem and Progenitor Cell Localization and Hypoxic Status in the Bone Marrow Microenvironment.</a> Nature Cell Biology. 15, 533-543 (2013).</li></ol>

There is nothing to disclose.

是什么成就？ 该协议采用延时多维成像流式细胞仪监测在小鼠颅骨骨髓移植纯化， 体外标记，移植HSPCs。单移植细胞的识别已经达到，这些都被监视的多的时间（hr）高精确度延长周期。呼吸引起的运动伪影被最小化，细胞已被多次重新制作映像在较长的时间过程。 什么是未来的发展方向？ 该技术的发展可以向回收实验的进展，以便为成像在多天里，在一个星期或更长时间，并利用气体麻醉剂如异氟醚的过程中，缩短和简化了回收过程的小鼠（注：回收实验要求严格无菌手术条件和适当的止痛药局）。开发elopment较大的彩色调色板体内染料以及造血细胞的有效的遗传标记也将促进多标记实验中，提供更深入骨髓内的细胞-细胞相互作用，并允许在未来更复杂的实验。此外，多个骨髓结构和间质成分的同时标示将提供正在发生的HSPC龛事件更完整的画面。的时间推移电影图像稳定性可以通过稳定的显微镜进一步以及减小了温度梯度的样品和购买后在通过使用图像配准算法可以提高预采集。 限制 ： 记载的技术提出了一些限制。小鼠以下注射麻醉剂的施用生存是不可预测的，这是很容易经历并发症取决于个别响应麻醉剂。通常，成像会话的时间越长，就越难是鼠标从麻醉中恢复，并且因此重要的是要仔细规划鼠标是否要回收，最好限制时间推移成像持续时间。使用注射麻醉药限制时间每个成像会议可以持续。虽然可以通过readministering较小剂量的麻醉剂，以延长麻醉时，难以执行这种精确和过量鼠标的过早终止的体内成像的最常见的原因之一。气体麻醉是毒性较低，并且允许更长的初始成像会话，但是快速的鼠标恢复后&gt; 2小时长的异氟醚的施用很少成功。该技术的另一个限制是，可以在多点延时成像而获得，由于需要快速获取图像的图像的分辨率有限。这一点，再加焦点漂移或现场跳（光盘上述ussed），可以使细胞追踪困难。 用另一种方法比较： HSPCs通常标有亲脂性染料一样，但尔和的DiI染料是等效的替代品和其他细胞的染料可以使用，只要足够亮，如在[16]点评。即使细胞的离体标记与DID已经显示出不影响造血干细胞的长期作用，它具有在细胞分裂稀释该DID的限制（或任何其它化学染料）。自造血干细胞在移植后的最初几天增殖，信号噪声比为这些细胞迅速劣化。细胞的遗传操作和荧光蛋白的表达的手段内源性标记是一种可行的替代方法，只要在荧光蛋白的表达在足够高的水平，以产生可检测的信号通过颅骨。有许多的替代技术部历史可iques到骨髓的空间内执行HSPCs成像，每个都有自己的优点和局限性。光纤为基础的成像器件允许插入骨髓重建的长骨[8]成像，而激光共聚焦成像下面的胫骨和牙釉质变薄了手术钻头手术暴露[12]所产生的HSPCs居住在详细图像长骨的周缘区域。然而这些方法都远比这里描述并且因此不允许重复成像会议集中于同一小鼠在相同的骨髓区域一个更侵入。此外，它是可能的，这些技术可能会影响观察到的给予在周围组织中广泛损害不可避免的响应的细胞。 HSPCs已经成像为很短的时间，通过放置在切下，裂隙股骨[11]盖玻片，这种恶劣的HSPCs的组织制备的但是影响不是CLEAr和技术被用来唯一地获取单个时间点的观测。固定骨头已经划分成连续切片，染色，所获得的图象重建为3D模型，从而提供优良的三维分辨率，尤其是当血管铸件使用[17]，以及最近的激光扫描仪（法援局）已被用来获得定量关于HSPCs 在原地措施，强调了免疫组化[18]，但无法提供有关的细胞行为的任何时间信息。在这种新技术所描述的技术提供了良好的分辨率在3D，足够时间采样的组合在4D监测细胞和它们相对非侵入性，因此提供了一个理想的方法，以达到长期监测HSPCs与骨髓互动微环境。

尽管已认识到了几十年3干细胞小生境和良好表征在许多组织中，如嗅球，肌纤维，毛囊膨出或CNS脑室下区4-7，（造血干细胞（HSCs）和骨髓微环境之间的相互作用BM）仍然很差，由于直接观察穿过骨单个细胞以及组织本身的极流体性质的难度理解。已经表明，通过一些功能性研究的基础上烧蚀或过度表达在任造血干细胞或微环境本身的基质细胞的特异性基因，即不同类型的细胞和调节信号的一个复杂的网络，负责调节静止之间微妙的平衡，增殖，扩张和造血干细胞1-2分化。造血干细胞和它们的生态位之间的串扰可以只通过直接观察来理解深度。这是ACHievable得益于先进的成像方案的发展，不仅在显微镜方面相结合的可利用的技术，也是试样夹持器的解决方案和采集软件。 中的小鼠颅盖骨的骨髓移植室的造血干细胞和祖细胞（HSPCs）的单细胞分辨率的实时成像显示，移植HSPCs定位中的SDF-1和VCAM-1-阳性血管8-9附近，并有更多的未成熟细胞从GFP阳性成骨细胞样细胞（Col2.3-GFP转基因小鼠品系，其中成骨细胞受限制的胶原蛋白1α启动子驱动GFP的表达），而它们的后代是更远端10 20微米-在15个被发现。类似的观察从解剖和股骨骨折的骨头11和薄tibeal骨头12获得。颅骨骨髓成像仍然是最侵入性的方式来实现HSPCs内日直视EIR原生微环境。 任何活体标本成像有一种内在的需要，以保持样品尽可能保持平稳，以避免任何不必要的文物由于样品运动。呼吸会导致鼠标头部的摆动运动，它需要避免成像颅骨骨髓的时候。传统的立体人，例如用于脑成像和电，不适合颅骨成像，因为它们阻碍了额骨。从使用，以尽量减少在现场的成像和电生理研究13窘迫鼠标座开始，2架组件开发，用一小片固定在鼠标的头部打造成像窗口连接到一个更大的手臂，确保稳抓稳了沉重的底板，它牢牢地固定到显微镜阶段。固定磁头片的小鼠的颅骨允许自由呼吸，同时仍然固定磁头就位并充分消除米ovements因呼吸。 A“锁和钥匙”的机制，连接头部件在支架体可在窗口的尺寸被最小化，以及定位的高精度化，因​​此，简化了外科手术，并且在合适的基片的插入阶段允许对在显微镜精确对准。准确地监测游动细胞，多点时间推移协议被设计为允许细胞的跟踪随时间的时间点之间的间隔5分钟。在这里，做标记的造血干细胞被注射到col2.3GFP osteoblasitc记者小鼠10,14和拍摄约20小时后。该设计并经过一段多个小时来执行快速多点延时的同一区域的成像所需要的成像设置的详细描述鼠标支架。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ketamine(Narketan 100mg/ml)</td>
			<td style="width:71px;">Vetoquinol</td>
			<td style="width:119px;">407575 0107 C</td>
			<td style="width:167px;">Other anesthetics may be used in place of this.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Medetomidine(Domitor 1mg/ml)</td>
			<td style="width:71px;">Elanco</td>
			<td style="width:119px;">134800-2</td>
			<td style="width:167px;">Other analgesics may b used in place of this.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Vibrant DiD cell labeling solution</td>
			<td style="width:71px;">Roche Products Ltd.</td>
			<td style="width:119px;">V22887</td>
			<td style="width:167px;">Other colors are available and may be used if required.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Lacrilube ophtalmic ointment</td>
			<td style="width:71px;">Allergan</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">avaliable by prescription by the local vet</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:216px;">Kemdent &#34;Diamond Carve&#34; glass ionomer cement</td>
			<td style="width:71px;">Associated Dental Products Ltd. via Kemdent</td>
			<td>SUN527</td>
			<td style="width:167px;">We use shade A3, but any shade of cement can be used.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PBS</td>
			<td style="width:71px;">multiple equivalent</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sterile water</td>
			<td style="width:71px;">multiple equivalent</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">Insulin syringes</td>
			<td style="width:71px;">Terumo Medical Corporation</td>
			<td style="width:119px;">SS10M3109</td>
			<td style="width:167px;">1mL, 31G</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Mouse restrainer</td>
			<td style="width:71px;">Harvard Apparatus</td>
			<td align="right" style="width:119px;">340031</td>
			<td style="width:167px;">Cone type (small)</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Autoclaved forceps and scissors</td>
			<td style="width:71px;">Agar Scientific</td>
			<td style="width:119px;">AGT577 
			AGT5034</td>
			<td style="width:167px;">Iris scissors - 90mm; Dumont HP tweezers (0.06mm tip)</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Weigh boat and cement stirrer</td>
			<td style="width:71px;">VWR</td>
			<td>611-1996/231-0370</td>
			<td style="width:167px;">5mL weigh-boat (black)/ Wooden tongue depressor</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Leica SP5 upright confocal microscope with motorized stage and two-photon laser</td>
			<td style="width:71px;">Leica Microsystems</td>
			<td>Not available</td>
			<td style="width:167px;">Call for quote</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">25x water dipping objective lens (N.A. = 0.95)</td>
			<td style="width:71px;">Leica Microsystems</td>
			<td align="right" style="width:119px;">11506323</td>
			<td style="width:167px;">HCX IRAPO L lens</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:216px;">Custom designed mouse holder</td>
			<td style="width:71px;">Imperial College Engineering Workshop</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">See Figure 1 for details</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Heating pad with rectal thermometer probe</td>
			<td style="width:71px;">BASi Instrumentation</td>
			<td style="width:119px;">FHC-40902/ FHC-40908/ FHC-4090502</td>
			<td style="width:167px;">Heat mat/ Control box/ Thermometer probe</td>
		</tr>
	</tbody>
</table>

in vivo 4-dimensional tracking of hematopoietic stem and progenitor cells in adult mouse calvarial bone marrow

通过静止和增殖，自我更新和分化产生子代之间的微妙平衡，造血​​干细胞（HSCs）维护所有成熟的血细胞谱系的营业额。导致特定的HSC命运的复数信号的协调依赖于造血干细胞和复杂的骨髓微环境，其仍知之甚少[1-2]之间的相互作用。 我们描述了如何通过新开发的试样保持器相结合的稳定的动物的定位与多步共聚焦和双光子中的体内成像技术，可以得到含有HSPCs高分辨率三维堆叠和其周围的壁龛，并监测它们随着时间的推移通过多点时间推移成像。高清晰度成像可检测离体标记的造血干细胞和祖细胞（HSPCs）驻留在骨髓内。此外，多点时间推移三维成像，邻btained更快的采集设置，提供了HSPC运动和HSPCs和基质细胞之间的相互交流准确的信息。 HSPCs相对于GFP阳性成骨细胞跟踪被示为这种方法的一个示例性应用。这种技术可以用来跟踪鼠标颅盖骨的骨髓空间内的感兴趣的任何适​​当标记的造血细胞或间质细胞。

做定制，高精度鼠标座，包括颅骨成像窗口让流式细胞仪纯化，标记注入致死剂量照射受体小鼠（ 图1A）HSPCs长时间成像。典型地，注射之间15的后 - 20000标记的细胞，8〜15细胞通常在翌日头骨的骨髓成像区域内辨认。这里，示出了如何获得HSPC含大约90骨髓领域的单细胞分辨率的三维堆叠- 120微米的厚度，（ 图1B）中，随后确定该HSPCs（ 图1C和电影）的4D时间推移电影。 不理想的结果，如果牙科用粘固剂不以最佳方式制备得到，例如，水可能泄漏从非密封区域，并且即使它可以从保持器的顶部加满，任何时间段，其中，透镜不沾入水导致黑色F<html> rames。有些漂移中的图像是不可避免由于鼠标保持器的材料的热膨胀，但它可以由不理想的水泥粘合性被加剧，导致小鼠的渐进脱离，并且由成像设置的扰动，从而导致突然的跳跃期间延时成像的焦点。在多点延时成像漂移也可以由不准确的阶段动作重新审视先前成像的位置时所引起的。在时间推移电影漂移和口吃的文物可以通过使用图像配准算法进行校正。 表1。激发和发射设置。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td style="width:204px;"> 色通道 </td><td style="width:213px;"> 激励 </td><td style="width:208px;"> 发射 </td></tr><tr><td style="width:204px;">绿色荧光蛋白</td>13px的;“&gt; 488 nm的</td><td style="width:208px;"> 500 - 530纳米</td></tr><tr><td style="width:204px;">自体荧光</td><td style="width:213px;"> 543纳米</td><td style="width:208px;"> 560 - 610纳米</td></tr><tr><td style="width:204px;"> DID </td><td style="width:213px;"> 633纳米</td><td style="width:208px;"> 640 - 700纳米</td></tr><tr><td style="width:204px;"> SHG </td><td style="width:213px;"> 840纳米（MP） </td><td style="width:208px;"> 400 - 450纳米</td></tr></tbody></table><img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51683/51683fig1highres.jpg" width="600" /> 图1： 在 HSPCs在小鼠体内颅骨四维成像。 （A）外形实验（B - C），得到的结果的例子（二）从3D堆叠二维切片（总深度为114微米），由骨胶原（白）含信号，做标记的细胞（红色） ，自体荧光CELLS（黄色）和成骨细胞（绿色）。比例尺：100微米（三）2D从4D定时影片呈现出做标记的造血干细胞（红色）在成骨细胞（绿色）附近迁移片。虚线突出显示单元的位移。比例尺为50μm（B）和100微米（℃）。注：从时间推移收购（C）得到的电影在视频文章显示<a href="https://www.jove.com/files/ftp_upload/51683/51683fig1large.jpg" target="_blank">，请点击这里查看该图的放大版本。</a>

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

造血干细胞和祖细胞（HSPCs）和骨髓龛之间的相互作用的性质知之甚少。定制的硬件修改和多步获取协议允许使用双光子共焦显微术图像体外骨髓区域内驻留标记HSPCs，跟踪相互作用和运动。

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

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

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Biology

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

13.7K 次观看.  Imperial College London. 造血干细胞和祖细胞（HSPCs）和骨髓龛之间的相互作用的性质知之甚少。定制的硬件修改和多步获取协议允许使用双光子共焦显微术图像体外骨髓区域内驻留标记HSPCs，跟踪相互作用和运动。 

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

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

归巢的骨髓造血细胞

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

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 ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

通过抑制Efflux泵,提高造血干细胞和造血细胞中线粒体膜电位流细胞测定的准确性

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

在维沃 小鼠头骨骨髓中巨细胞和前列腺的双光子成像

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

使用细胞外通量分析仪评估小鼠造血干细胞和原始祖细胞中的细胞生物能量学

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...