这项工作得到了德克萨斯州骨病计划奖，Caroline Wiess法律基金奖和美国国立卫生研究院NIAMS的支持，奖项编号为R01 AR072018，R21 AG064345，R01 CA221946至D.P。 我们感谢M.E. Dickinson和T.J. Vadakkan在BCM Optical Imaging and Vital Microscopy Core 以及BCM Advanced Technology Cores的资助下，由NIH资助（AI036211，CA125123和DK056338）。
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所有小鼠都保持在无病原体的条件下，所有程序均由贝勒医学院的机构动物护理和使用委员会（IACUC）批准。
1. 鼠标准备
<ol><li>将Mx1-Cre17和Rosa26-loxP-stop-loxP-tdTomato（Tom）18只小鼠（从杰克逊实验室购买）与αSMA-GFP小鼠（由Ivo Kalajzic博士和Henry Kronenberg博士提供）杂交，生成Mx1-Cre + Rosa26-Tomato + α SMA-GFP+（Mx1 / Tomato / αSMA-GFP）报告小鼠（图2A）。</li>
	<li>对于 Mx1-Cre诱导，每隔一天向小鼠注射25mg / kg pIpC，持续10天。</li>
	<li>在静脉移植来自野生型C57BL / 6 小鼠（WT-BMT）的1 x 10 6全骨髓单核细胞之前1天用9.5 Gy照射小鼠19。</li>
	<li>在恢复6至8周后，对小鼠进行如下所述的骨膜细胞迁移方案的体内成像。 注意： Mx1-Cre小鼠的辐照和WT-BMT对于消除可能对损伤有反应的内源性 Mx1标记造血细胞是必要的。照射后六到八周，宿主的造血细胞计数为<5%，因此可以进行成像19。</li>
</ol>2. 鼠标约束和成像支架
<ol><li>使用50 mL锥形管创建鼠标约束（图1A）。<ol>
<li>使用尖头剪刀，在圆锥体的底部戳一个洞。从底部孔切割到锥形的顶部（切割侧将是约束的顶部）。</li>
		<li>切口与前一个切口平行约4-5厘米长，从约束装置顶部向下一半（约2厘米）。去除这一部分的圆锥形并平滑边缘是必要的。</li>
	</ol>
</li>
	<li>使用可调节角度板，0.5"光学柱和0.5"柱，0.1875"六角的直角夹具构建锥形管成像支架（图1B 和 材料表）。<ol>
<li>将角板调整为 35° 角。在角板的左侧，将光学柱从板的背面拧入第二个孔中。</li>
		<li>在可调节板的右侧，将弹簧加载的0.1875"六角锁定0.25"指旋螺钉从板上的前孔和最后一个孔插入第二个孔中。直角夹具将与光学柱一起使用，以将锥形鼠标约束固定到位。</li>
	</ol>
</li>
</ol>3. 术前外科手术
<ol><li>小鼠麻醉<ol>
<li>对于疼痛管理，在手术前30-60分钟皮下注射缓释丁丙诺啡（1mg / kg BW）。</li>
		<li>使用含有37.5mg / mL氯胺酮，1.9mg / mL木拉嗪和0.37mg / mL乙丙嗪的啮齿动物IIICCM组合（组合III）麻醉小鼠。小鼠的适当剂量为每1公斤体重0.75-1.50毫升。剂量将持续30-45分钟。</li>
		<li>麻醉小鼠后，用无菌棉签将眼药膏涂抹在眼睛上，以防止眼睛脱水。</li>
		<li>对于长期影像学检查（>45分钟），再次进行麻醉以保持适当的麻醉深度，如下所示。<ol>
<li>准备一个1 mL注射器，其中含有足够量的组合III，以在计划的成像持续时间内保持麻醉（每30分钟额外镇静0.75 mL / kg BW）。将带有12英寸管的25 G蝴蝶针输液装置连接到注射器上，然后将麻醉剂推入管路和针头。</li>
			<li>将蝴蝶针插入腹腔内，然后将针头粘到位。 注意：这就是在成像期间施用附加组合III的方式，这样就不需要移动动物或破坏视平面。</li>
		</ol>
</li>
		<li>在整个过程中，通过保持在37°C的加热垫提供热支持。对于长时间的影像学检查（>1小时），需要额外的液体支持。 在影像学检查期间，可向 IP 患者提供补充液体和额外的 Combo III，或皮下注射 33 mL/kg 生理盐水 （0.9 NaCl）。</li>
	</ol>
</li>
	<li>小鼠约束和手术部位的准备<ol>
<li>使用剪刀尽可能多地去除头顶的头发。将脱毛膏涂抹在剃须区域，以去除手术部位的任何剩余毛发。</li>
		<li>确保鼠标因对脚趾捏合缺乏反应而完全麻醉。15分钟后，如果小鼠未完全麻醉，则给予第二小剂量的组合III（<0.75mL / kg BW）。</li>
		<li>首先将鼠标放在约束尾部，向后滑动足够远，使鼻子略微悬挂在圆锥形的边缘上。</li>
		<li>使用医用胶带将前爪粘在锥形管的底部。</li>
		<li>将带鼠标的锥形管放在设置为35°角的成像支架上，并使用直角夹具固定（图1C）。</li>
		<li>一旦小鼠固定在成像支架中，用β定磨砂膏和70%异丙醇交替擦洗3次手术部位。再次，检查动物是否完全镇静。</li>
		<li>将鼠标移动到干净的吸水垫上，并在鼠标上放置一个悬垂物以创建一个大的无菌区域。</li>
	</ol>
</li>
</ol>4. 外科手术
<ol><li>开口切口<ol>
<li>使用细尖镊子，立即将皮肤从内侧到右耳。用显微切割剪刀，切开被镊子固定的皮肤。 注意：使用上述方法启动切口会产生一个圆形切口，更适合在手术后缝合。</li>
		<li>从初始切口开始向左耳（<1厘米）做一个横向切口。 从初始切口开始，再切口朝向动物的鼻子（右眼后约2-3毫米）。</li>
		<li>使用镊子和剪刀将皮肤与骨膜分开，轻轻切开结缔组织，将皮肤与骨膜隔开。</li>
		<li>使用无菌棉签，将一些眼药膏涂抹在皮瓣的顶部。 轻轻拿起皮瓣，将其折叠在左眼上。矢状缝合线和冠状缝合线的交点应清晰地暴露（图1E）。 注意：眼科软膏用于保持皮瓣到位。</li>
		<li>用无菌PBS冲洗开放表面，以清洁任何血液和残留头发的区域。 注意：如有必要，请使用细尖镊子去除冲洗后残留的毛发，但注意不要损坏骨膜。</li>
	</ol>
</li>
	<li>微骨折<ol>
<li>为了在颅骨上产生微骨折，请取出29 G胰岛素注射器的柱塞，并切割注射器的后端，大约300-400 uL标记。 注意：此步骤使得在产生伤害时更容易在解剖镜下操作。</li>
		<li>将斜角的尖端（一到两个针宽）从冠状缝合线朝向鼻子，将一到两个针宽放在矢状缝合线的右侧（图1E）。</li>
		<li>顺时针轻轻旋转注射器，然后逆时针旋转几次。 注意：在年轻小鼠中，需要非常少量的向下压力来产生圆形微骨折。注意不要让针头穿透大脑，因为这会导致出血并干扰成像。</li>
		<li>使用27 G针将微骨折扩大至~0.4 mm。如果骨碎片仍留在微骨折中，请用PBS冲洗该区域。如果骨碎片仍然留在微骨折中，请使用29 G针轻轻地将其舀出。</li>
		<li>重复步骤4.2.1-4.2.4，在矢状缝合线左侧造成损伤。 注意：此时，有两种选择：1）立即进行CCL5治疗（第4.3节）和成像（第5节）;或2）在第6节中的术后程序后关闭手术部位。24小时后，再次麻醉小鼠，重新打开手术视力，用CCL5治疗损伤（第4.3节），并进行活体成像（第5节）和术后护理（第6节）。</li>
	</ol>
</li>
	<li>CCL5 处理<ol>
<li>从兰特斯（100 ng / mL）的储备中，使用基底膜基质（材料表）制备10 ng / mL的溶液。将这种溶液保持在冰上，以保持基质为液体形式。</li>
		<li>使用2-20μL移液器用2μLCCL5（10ng / μL）治疗每个损伤（该移液器吸头具有更大的直径，使用粘性液体时更有效）。让基质凝固。</li>
		<li>一旦基质凝固，用皮瓣轻轻覆盖颅骨，以确保组织不会脱水。在成像前孵育1小时。</li>
	</ol>
</li>
</ol>5. 活体成像
<ol><li>显微镜设置<ol>
<li>打开多光子激光器（飞秒钛蓝宝石激光器）。将波长设置为 880 nm，并调整骨骼二次谐波发生 （SHG） 成像 （440 nm） 的功率。</li>
		<li>打开488 nm和561 nm固体激光器，分别激发GFP和tdTomato表达细胞。</li>
		<li>打开 PMT（光电倍增管）探测器，用于二次谐波生成（405–455 nm 检测）和 GFP（505–550 nm 检测）信号。打开 HyD 3 检测器以检测番茄（590–620 nm 检测）信号。</li>
	</ol>
</li>
	<li>安装<ol>
<li>在将鼠标移动到示波器之前，请轻轻按压鼠标后脑勺来检查颅骨的视觉平面。如果平面不水平，请通过向左或向右轻轻旋转鼠标约束来调整位置。 注：这是确保足够图像质量的关键步骤。</li>
		<li>将无菌的2%甲基纤维素在水中（w / v）中涂抹，以覆盖整个颅骨和皮瓣，并防止组织干燥。</li>
		<li>将鼠标放在XYZ轴电动显微镜载物台上（图1G）。使用落射荧光，对齐鼠标，使其1）面向显微镜，2）冠状和矢状缝合线的交点是载物台的中心参考点。</li>
		<li>将玻璃盖放在成像区域并仔细检查对齐情况（图1H）。</li>
	</ol>
</li>
	<li>延时成像<ol>
<li>使用低放大倍率镜头（25倍水浸物镜，NA = 0.95）扫描钙质。获取矢状肌和冠状缝合线交叉的参考图像。</li>
		<li>使用来自细胞的SHG和荧光信号，定位每个损伤视力并记录XYZ坐标以及与矢状肌和冠状缝合线的距离（图2B）。</li>
		<li>在冠状或矢状缝合线上选择一个位置进行长期成像。在成像过程中从参考中获取此区域的详细 Z 堆栈图像。 注意：由于缝合线代表已知的P-SSC生态位，这是细胞响应颅骨损伤而迁移的地方。</li>
		<li>使用延时软件设置每1分钟记录一张图像至少1小时。在快照之间的时间内，将当前视野与初始视野进行比较。如果它们不同，请使用 Z 轴图像来确定需要调整视野的方向。</li>
	</ol>
</li>
</ol>6. 术后动物护理
<ol><li>成像后，用无菌PBS冲洗暴露的钙质以除去甲基纤维素。</li>
	<li>要关闭切口，轻轻地将皮瓣放在颅骨上。使用无菌棉签去除残留的眼科软膏并干燥切口部位。</li>
	<li>采用单丝尼龙5-0尺寸不可吸收缝合线，附有C-1反向切割针，闭合手术部位。使用无菌棉签将三重抗生素软膏涂抹在封闭的切口上。 注意：通过高质量的缝合技术和最小的出血减少疤痕。</li>
	<li>将鼠标放在干净的笼子中，放在温暖的表面上，每15分钟检查一次，直到达到胸骨卧位。</li>
	<li>术后72小时给予第二剂缓释丁丙诺啡。</li>
	<li>每天监测蜷缩行为、褶皱的皮毛和/或缺乏行走，直到缝合线被移除（约 1 周）。</li>
</ol>

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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=Identification+of+Functionally+Distinct+Mx1+alphaSMA++Periosteal+Skeletal+Stem+Cells.">Identification of Functionally Distinct Mx1+alphaSMA+ Periosteal Skeletal Stem Cells.</a> Cell Stem Cell. 25 (6), 784-796 (2019).</li><li>Deveza, L., Ortinau, L., Lei, K., Park, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+gene+expression+identifies+distinct+molecular+signatures+of+bone+marrow-+and+periosteal-skeletal+stem/progenitor+cells.">Comparative analysis of gene expression identifies distinct molecular signatures of bone marrow- and periosteal-skeletal stem/progenitor cells.</a> PLoS One. 13 (1), 0190909 (2018).</li><li>Schindeler, A., McDonald, M. M., Bokko, P., Little, D. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leptin-receptor-expressing+mesenchymal+stromal+cells+represent+the+main+source+of+bone+formed+by+adult+bone+marrow.">Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow.</a> Cell Stem Cell. 15 (2), 154-168 (2014).</li><li>Grcevic, D., 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=In+vivo+fate+mapping+identifies+mesenchymal+progenitor+cells.">In vivo fate mapping identifies mesenchymal progenitor cells.</a> Stem Cells. 30 (2), 187-196 (2012).</li><li>Kuhn, R., Schwenk, F., Aguet, M., Rajewsky, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+targeting+in+mice.">Inducible gene targeting in mice.</a> Science. 269 (5229), 1427-1429 (1995).</li><li>Srinivas, S., 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=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Developmental Biology. 1, 4 (2001).</li><li>Park, D., 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=Endogenous+bone+marrow+MSCs+are+dynamic,+fate-restricted+participants+in+bone+maintenance+and+regeneration.">Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration.</a> Cell Stem Cell. 10 (3), 259-272 (2012).</li><li>Nitzsche, F., 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=Concise+Review:+MSC+Adhesion+Cascade-Insights+into+Homing+and+Transendothelial+Migration.">Concise Review: MSC Adhesion Cascade-Insights into Homing and Transendothelial Migration.</a> Stem Cells. 35 (6), 1446-1460 (2017).</li><li>Adler, J., Pagakis, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+image+distortions+due+to+temperature-related+microscope+stage+drift.">Reducing image distortions due to temperature-related microscope stage drift.</a> Journal of Microscopy. 210, 131-137 (2003).</li><li>Roberts, S. J., Ke, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anabolic+Strategies+to+Augment+Bone+Fracture+Healing.">Anabolic Strategies to Augment Bone Fracture Healing.</a> Current Osteoporosis Reports. 16 (3), 289-298 (2018).</li></ol>

L.C.O.和D.P.公开了一项正在申请的专利，名为"骨骼修复中的骨膜骨骼干细胞"（BAYM.P0264US.P1）。其余作者声明没有竞争利益。

在骨愈合过程中，骨膜细胞是损伤愈伤组织内新分化的软骨细胞和成骨细胞的主要来源3。与骨髓类似，在骨膜中也发现了成骨/软骨SSC4，5，6。评估内源性P-SSC功能特征在技术上具有挑战性。通常，SSC的迁移动力学是在体外评估的，这使得对其相应体内系统的解释具有挑战性。由于活体动物活体活体显微镜检?...

骨折修复是一种动态的多细胞过程，与胚胎骨骼发育和重塑不同。在此过程中，从受损组织诱导损伤信号后，骨骼干/祖细胞的快速募集、增殖和随后分化，这些都对骨折的整体稳定性和固定至关重要1。特别是，骨折愈合的早期阶段需要软愈伤组织形成，这主要归因于骨膜常驻细胞2。当骨骼受伤时，骨膜细胞的一个亚群会迅速做出反应，并导致愈伤组织内新分化的软骨中间体和成骨细胞3，暗示骨膜内存在明显的骨骼干/祖细胞群。因此，骨膜驻留骨骼干细胞（SSC）的鉴定和功能表征是治疗退行性骨病和骨缺损的一种有前途的治疗方法4。
SSC被认为存在于多个组织位置，包括骨髓。与骨髓类似，在骨膜中也发现了成骨/软骨SSC4，5，6。 这些骨膜 SSC （P-SSC） 可在胎儿骨骼发育过程中使用早期间充质谱系标志物（即 Prx1-Cre5、Ctsk-Cre7 和 Axin2-CreER8）进行标记4，7，8，9，10。这些单一遗传谱系追踪模型的一个显着局限性是标记细胞群中存在实质性的异质性。此外，它们无法将标记的SSC与其体内的后代区分开来。为了解决这一限制，我们最近开发了一种双报告小鼠（Mx1-Cre+ Rosa26-Tomato+α SMA-GFP+），以清晰地可视化骨髓SSC（BM-SSC）中的P-SSC11。通过该模型，已经确定P-SSC以Mx1 + αSMA +双重标记标记标记，而更多分化的Mx1 +细胞驻留在骨髓，骨内和骨膜表面以及皮质和小梁骨11，12。
已知多种细胞因子和生长因子可调节骨重塑和修复，并且已经过测试，以增强关键节段缺损中的骨骼修复1，13。然而，由于通过破坏骨室的物理屏障产生的损伤模型的细胞复杂性，这些分子对内源性P-SSC迁移和愈合期间激活的直接影响尚不清楚。SSC的功能特征和迁移动力学通常在体外通过进行转孔或划痕测定来评估，并结合已知可诱导其他细胞群迁移的细胞因子或生长因子。因此，解释这些体外实验的结果以应用于其相应的体内系统是具有挑战性的。目前，骨骼干/祖细胞迁移的体内评估通常不是实时观察到的;相反，它是在骨折后的固定时间点测量的5，7，14，15，16。
这种方法的局限性是没有在单细胞水平上评估迁移;相反，它是通过细胞群的变化来测量的。由于活体动物活体显微镜检查和额外报告小鼠的产生的最新进展，现在可以对单个细胞进行体内跟踪。使用活体动物活体显微镜，我们观察到Mx1 / Tomato / αSMA-GFP小鼠在受伤后24-48小时内P-SSC从颅骨缝合龛位到骨损伤的不同迁移。
CCL5/CCR5最近被确定为一种调节机制，在早期损伤反应期间影响P-SSC的招募和激活。有趣的是，没有检测到大量的P-SSC迁移以响应实时损伤。然而，用CCL5治疗损伤会产生P-SSC的稳健，方向上不同的迁移，可以实时捕获。因此，该方案的目的是提供一种详细的方法，用于在用CCL5处理后实时记录P-SSC的体内迁移。

<table border="0" cellpadding="0" cellspacing="0" style="width:720px;" width="719">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">&#189;&#8221; optical post</td>
			<td style="width:175px;">ThorLabs</td>
			<td style="width:135px;">TR2</td>
			<td style="width:247px;">For imaging mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">1 mL syringe</td>
			<td style="width:175px;">BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">27G needle</td>
			<td style="width:175px;">BD PercisionGlide</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">29G insulin syringe</td>
			<td style="width:175px;">McKesson</td>
			<td>102-SN05C905P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">50 mL conicol tube</td>
			<td style="width:175px;">Falcon</td>
			<td>352098</td>
			<td>For mouse restraint</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Adjustable angle plate</td>
			<td style="width:175px;">Renishaw</td>
			<td>R-PCA-5023-50-20</td>
			<td>For imaging mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Alcohol wipes</td>
			<td style="width:175px;">Coviden</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">betadine surgical scrub</td>
			<td style="width:175px;">Henry Schen</td>
			<td>67618-151-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Buprenorphine SR-LAB</td>
			<td style="width:175px;">ZooPharm</td>
			<td></td>
			<td>1mg/mL Sustained Release</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Combo III</td>
			<td style="width:175px;">Obtained from staff veterinarian</td>
			<td>N/A</td>
			<td style="width:247px;">37.6 mg/mL Ketamine; 1.9 mg/mL Xylazine; 0.37 mg/mL Acepronazine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Coverslip</td>
			<td style="width:175px;">Fisher</td>
			<td>12-545-87</td>
			<td>24 x 40 premium superslip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Fine tip forcepts</td>
			<td style="width:175px;">FST</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Ketamine</td>
			<td style="width:175px;">KetaVed</td>
			<td>50989-161-06</td>
			<td style="width:247px;">100 mg/mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Leica TCS SP8MP with DM6000CFS</td>
			<td style="width:175px;">Leica Microsystems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Matrigel</td>
			<td style="width:175px;">R &#38; D Systems</td>
			<td>344500101</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:164px;">Medical tape</td>
			<td style="width:175px;">McKesson</td>
			<td>100199</td>
			<td>3&#34; x 10 yds (7.6 cm x 9.1 m)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Methocellulose</td>
			<td style="width:175px;">Electron Microscopy Sciences</td>
			<td>19560</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Microdissection scissors</td>
			<td style="width:175px;">FST</td>
			<td>1456-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Motorized stage</td>
			<td style="width:175px;">Anaheim automation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Needle holder</td>
			<td style="width:175px;">FST</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Nonabsorbable sutures</td>
			<td style="width:175px;">McKesson</td>
			<td>S913BX</td>
			<td style="width:247px;">monofilament nylon 5-0 nonabsorbable sutures with attached C-1 reverse cutting needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Opthalmic ointment</td>
			<td style="width:175px;">Rugby</td>
			<td>0536-1086-91</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">RANTES</td>
			<td style="width:175px;">Biolegend</td>
			<td>594202</td>
			<td>10 &#181;g/50 &#181;L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Right-angle clamp for &#189;&#8221; post, 3/16&#8221; Hex</td>
			<td style="width:175px;">ThorLabs</td>
			<td>RA90</td>
			<td>For imaging mount</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Spring-loaded 3/16&#8221; Hex-locking &#188;&#8221; thumbscrew</td>
			<td style="width:175px;">ThorLabs</td>
			<td>TS25H</td>
			<td>For imaging mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Sterile cotton swabs</td>
			<td style="width:175px;">Henry Schen</td>
			<td>100-9249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Sterile DPBS (1x)</td>
			<td style="width:175px;">Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:164px;">Sterile drapes</td>
			<td style="width:175px;">McKessen</td>
			<td>25-517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Surgical gloves</td>
			<td style="width:175px;">McKessen</td>
			<td>3158VA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Triple antibiotic ointment</td>
			<td style="width:175px;">Taro Pharmaceuticals U.s.a., Inc.</td>
			<td>51672-2120-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Vacutainer blood collection set</td>
			<td style="width:175px;">BD</td>
			<td style="width:135px;">REF 367298</td>
			<td style="width:247px;">25G butterfly needle infusion set with 12&#34; tubing</td>
		</tr>
	</tbody>
</table>

real-time imaging of ccl5-induced migration of periosteal skeletal stem cells in mice

骨膜骨骼干细胞（P-SSC）对于终身骨骼维护和修复至关重要，使其成为开发增强骨折愈合的疗法的理想焦点。 骨膜细胞迅速迁移到损伤处，为骨折愈合提供新的软骨细胞和成骨细胞。传统上，细胞因子诱导细胞迁移的功效仅在体外通过进行转孔或划痕测定进行。随着使用多光子激发的活体显微镜检查的进步，最近发现1）P-SSC表达迁移基因CCR5和2）使用称为CCL5的CCR5配体处理改善了骨折愈合和P-SSC响应CCL5的迁移。这些结果是实时捕获的。这里描述的是一种方案，用于可视化P-SSC从颅骨架骨骼干细胞（SSC）生态位迁移到CCL5治疗后的损伤。该协议详细介绍了小鼠约束装置和成像支架的构造，小鼠颅骨的手术准备，颅骨缺陷的诱导以及延时成像的采集。

骨骼祖细胞被认为具有迁移或循环潜力20。最近，生成了Mx1-Cre+ Rosa26-Tomato+α SMA-GFP+（Mx1/Tomato/αSMA-GFP）报告小鼠，其中P-SSC用Mx1 + α SMA +双重标记标记（图2A，B）。Mx1+ α SMA+ P-SSC在损伤后24-48小时内从缝合间充质中大量迁移<sup cl...

Watch this Scientific Journal Video about Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice at JoVE.com

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

该协议描述了使用活体动物活体显微镜实时检测CCL5介导的骨膜骨骼干细胞迁移。

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

real-time-imaging-ccl5-induced-migration-periosteal-skeletal-stem

Research

JoVE Journal

Developmental Biology

2.2K 次观看.  Baylor College of Medicine. 该协议描述了使用活体动物活体显微镜实时检测CCL5介导的骨膜骨骼干细胞迁移。

视频: Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

多光子时间推移成像实时可视化发展： 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

在免疫缺陷小鼠中表演人类骨骼肌异种移植物

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...