这项工作得到了美国国立卫生研究院拨款R01-HD093827和R03-HD090304（AJT）的支持。

所有动物程序均根据指南进行，并得到康奈尔大学机构动物护理和使用委员会 （#2002-0095） 的批准。本研究使用 8-10 周龄的 AcroSensE 小鼠2 。有关AcroSensE鼠标可用性的信息请求可以提交给通讯作者。
1.精子收集和清洗
<ol><li>从AcroSensE小鼠中收集附睾尾精子（有关此程序的更多详细信息在11中提供）。在37°C下含有0.5mL改良Whitten培养基（MW）的3.5cm组织培养皿中允许15分钟的游出程序。 培养皿应被覆盖以减少MW的蒸发，并且应倾斜，使培养基有足够的深度来覆盖附睾尾部。 注意：改良的Whitten培养基（分子量）包括22mM HEPES，1.2mM MgCl 2,100mM NaCl，4.7mM KCl，1mM丙酮酸，4.8mM乳酸hemi-Ca2+盐，pH 7.35。葡萄糖（5.5mM），NaHCO3（10mM）和2-羟丙基环糊精（CD;3mM）根据需要补充（见材料表）以诱导获能，并且可以针对特定实验修改浓度。此外，可以使用替代甾醇受体代替克罗恩病（例如，牛血清白蛋白或高密度脂蛋白）。</li>
	<li>使用细镊子，从游泳收集皿中取出任何附睾组织。将含有精子的培养基转移到15mL锥形管中，并在室温下将悬浮液在摆动桶转子中以100× g 离心1分钟。使用塑料移液管，从在此步骤中沉淀的任何大组织碎片中取出含有精子的MW上清液。</li>
	<li>通过在37°C下加入MW，使含有精子的MW上清液的总体积达到3mL。 在圆底管中以400× g 离心8分钟（室温下）。从松散颗粒的精子中慢慢除去MW的上清液。</li>
	<li>如果可行，精子应该看起来像一个“蓬松”的颗粒。 通过 用大口径移液管研磨或 轻 轻手动敲击管子（“手指轻弹”）轻轻重悬精子。重新悬浮均匀后，将它们转移到新的圆底管中。使用 10-20 μL 精子悬浮液来计数和评估活力。在MW中稀释精子以供使用。 注意：对于大多数涉及小鼠精子获能的实验，使用5-1000万精子/ mL MW的最终浓度。这里描述的成像方案不需要精子的获能，尽管精子必须获得经历生理相关的顶体胞吐作用的能力。人们可以研究病理性胞吐作用或通过钙离子载体等试剂诱导的胞吐作用，而不会使精子具有能力。此外，人们可以探索非容量细胞中顶体钙响应各种刺激的潜在变化。</li>
	<li>在收集后3-5小时内使用精子，使时间尽可能短，并在实验试验中保持一致。</li>
	<li>使用MW培养基在37°C下执行收集和洗涤的所有步骤，采用尽量减少膜损伤的方法。这包括使用大口径塑料移液器或大孔径移液器吸头，建议在手术的所有阶段使用。</li>
</ol>2. 用于成像的盖玻片培养皿的聚-D-赖氨酸涂层
注意：聚-D-赖氨酸 （PDL） 培养皿应在每次实验前新鲜制备。
<ol><li>在盖玻片培养皿的中心分配0.5μL PDL液滴（0.5mg / mL，参见 材料表）。</li>
	<li>使用10μL刻度吸头，将PDL液滴涂抹在盖玻片上（纵横交错的图案，如 图1B所示）。</li>
	<li>在37°C下干燥10分钟。将涂有PDL的培养皿盖上盖子并保持在37°C直至使用。</li>
</ol>3.毛细管拉动，用于膨化
<ol><li>使用如下设置拉动硼硅酸盐玻璃毛细管（外径，2 mm，内径，1.56 mm，参见 材料表）- 热量：330，拉力：250，速度：250和时间：70。此步骤应针对使用的特定拉拔器进行优化。</li>
	<li>在每次实验之前，用含有3-5倍兴奋剂浓度的刺激溶液加载单个毛细管（以解释溶液在膨化时的快速扩散）。填充前，用细镊子在距末端约 1-2 毫米处掰开毛细管尖端。这应该产生 ∼5 μm 的开口直径。不需要热抛光。</li>
	<li>使用薄塑料移液管，将刺激溶液缓慢注入毛细管中，直到其长度的四分之三充满。</li>
	<li>通过轻弹动毛细管去除填充过程中形成的任何气泡。</li>
	<li>将填充的毛细管安装在显微操纵器上（参见 材料表）。</li>
</ol>4.准备为膨化提供刺激
注意： 为了最大限度地降低用户受伤的风险，在膨化程序操作过程中应佩戴护目镜。
<ol><li>设置单电池输送系统设置，以在 5 psi 下提供 10 秒脉冲。</li>
	<li>将硼硅酸盐毛细管连接到单细胞输送系统移液器支架。确保密封严密。</li>
	<li>在观察毛细管尖端时，在 20 psi 下短吸 2 秒，以确保溶液确实通过尖端末端分配（尖端末端应明显有一小滴）。</li>
</ol>5. 显微镜和图像采集
注意：有多种品牌的显微镜可供选择，可以使用;但是，最低帧速率为 3 帧/秒是可取的。关于温度和环境控制，请注意，应确认培养皿中介质的实际温度，例如使用非接触式红外测温仪。
<ol><li>将80μL稀释的精子加入聚D-赖氨酸包被的35mm盖玻片培养皿的中心。对于大多数涉及小鼠精子获能的实验，使用5-1000万精子/mL的最终浓度。</li>
	<li>向精子中缓慢加入补充有10mM CaCl2的3mL温（37°C）MW基础培养基。 注意：人们可以在室温下进行实验以减缓细胞过程，但重要的是要注意，由于获能是由脂质交换以及微观和宏观结构域的流动性介导的，因此必须记住，生理相关的获能和顶体胞吐作用是温度依赖性过程。</li>
	<li>将精子与精子一起安装在显微镜上（预热至37°C）。</li>
	<li>使用 488 nm 激光线激发 GCaMP3，并使用 505-550 nm 带通 （BP） 滤光片查看。使用 555 nm 激光线激发 mCherry，并使用 575-615 nm BP 滤光片查看。</li>
	<li>使用显微操纵器（建议将机械手连接到设置显微镜的气台上），将毛细管的尖端放置在感兴趣的细胞平面上方约100μm和5-10μm。 注意：刺激溶液以正常浓度的 3-5 倍制备，以补偿毛细管和细胞之间体积中发生的快速扩散。</li>
	<li>在显微镜上开始成像序列。</li>
	<li>以高帧速率（优选 >10 帧/秒）对精子细胞进行成像。在这里，使用了共振扫描仪，能够以 33 毫秒/帧的速度成像。</li>
	<li>在显微镜上开始图像采集10秒后，激活单细胞递送系统以启动10秒的兴奋剂。</li>
	<li>成像细胞持续时间为10-15分钟。</li>
	<li>使用单细胞递送系统，根据 图1B中提供的位置对单个培养皿中的多个细胞进行成像。 注意：在显微镜下未经处理/未受刺激的精子应大部分呈红色，绿色信号强度较低。精子头应附着在盖玻片上，活精子可以通过其移动的尾巴来识别。在经历AE的精子中，GCaMP3绿色信号最初会增加，如果AE完成，mCherry红色信号和GCaMP3绿色信号都会消失，如 图2所示。mCherry荧光的减少为AE开始的时间提供了更具体的指标，因为Ca2+ 浓度的变化将不断导致GCaMP3荧光强度的变化。</li>
</ol>6. 图像和数据分析
注意：离线图像分析是使用 ImageJ 进行的（参见 材料表）。此前，在导致 AE 的过程中报道了几个中间步骤，包括顶体钙升高 （ACR） 和全膜融合。后者导致 mCherry 荧光的损失，因此代表完全 AE。在一些细胞中，当使用电化学方法进行胞吐作用研究时，观察到类似于尖峰前足事件 （PSF） 的信号（有关详细信息，请参见2）。有几种可选方法可用于分析 AcroSensE 原始数据，以量化 ACR、AE 及其中间步骤。下面介绍一些基本的分析方法。
<ol><li>根据特定的显微镜系统文件扩展名，使用分割通道工具（Image > Colors > Split Channels）为 GCaMP3 和 mCherry 通道生成单独的窗口。</li>
	<li>使用“同步窗口”工具（分析>工具>同步窗口）同步各个窗口。</li>
	<li>在红色通道中选择精子头部周围的感兴趣区域（ROI），以包括顶体区域（图3A，B）。同步工具允许在绿色通道上自动应用相同的区域选择，其中精子仍然太暗而无法看到。</li>
	<li>选择“Z Profiler”插件（Z Profiler > Stacks > Plugins ）或“Time Series Analyzer”（Plugins > Stacks > Time Series Analyzer），以绘制两个通道中每个通道的荧光强度变化随帧数/时间的变化。</li>
	<li>将数据复制到电子表格软件（例如 Microsoft Excel）以进行绘图和进一步分析。 注意：将数据复制到电子表格后，可以提取多个参数进行分析，包括以下参数，如 图 4 所示。<ol><li>熔孔 （FP） 开始时间：测量从时间点 0 到 GCaMP3 信号开始上升的时间点的时间。该参数表示刺激和精子ACR反应之间的延迟。</li>
		<li>AE 开始时间：测量从时间点 0 到 mCherry 信号开始衰减的时间点的时间。该参数表示刺激和精子AE反应之间的延迟。该参数还可用于计算 FP 形成和 AE 响应之间的延迟。</li>
		<li>峰值FP幅度：测量荧光信号从基底到GCaMP3信号上升的峰值。此参数指示 ACR 响应的总增加量。</li>
		<li>mCherry 的损耗率：通过对 mCherry 信号损耗部分的线性或指数拟合来确定此参数（如 图 4B 中的“斜率”所示）。 注意：或者，通过残差 （R） 信号法确定不同时间点的衰减率（例如，R60：持续时间（以秒为单位）到距 mCherry 信号基线 60% 的残余信号）。该参数可用于量化顶体内容物在 AE 时分散的速率。</li>
		<li>mCherry 丢失的信号降低：确定基线 mCherry 荧光强度与 AE 后信号之间的幅度差异。该参数表示在 AE 上分散的顶体内容物的总量。</li>
		<li>将红色和绿色通道的荧光（F）变化绘制为原始数据，或通过初始荧光（F0）对其进行归一化，并将其表示为ΔF / F0。后者可以更好地可视化单个图上红色和绿色的变化（例如，参见 图3D 和 图3H）。 注意：最后，可以比较不同条件下的不同测量参数，以确定顶体钙浓度变化与AE之间的关系。有关各种实验条件比较的示例，请参阅参考文献2。</li>
	</ol></li>
</ol>

将成像与兴奋剂递送系统相结合以研究活体小鼠精子

<ol>
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没有作者有与这项工作相关的利益冲突。

在这里，描述了一种基于显微镜的方法，利用新生成的 AcroSensE 小鼠模型进行实时单细胞监测和分析顶体钙动力学与导致 AE 的中间步骤之间的相互作用。与现成的遗传方法（例如与其他小鼠遗传模型杂交或基因编辑）一起，该模型和方法提供了一个强大的系统来研究参与与获能、AE 和受精相关的精子信号通路的各种成分和途径的作用。
关键步骤 在所有精子处?...

精子在称为获能1 的过程中获得受精能力。这个过程的一个终点是精子获得经历AE的能力。超过二十年的数据支持哺乳动物精子中存在复杂的多步骤AE模型（总结于2,3）。然而，研究活精子中的AE具有挑战性，目前可用的以足够分辨率监测这一过程的方法很麻烦，需要多个制备步骤4，仅限于检测AE的最后一步（例如，使用PNA5），仅限于测量胞质钙的变化（与顶体钙动力学相反）， 或仅限于细胞溶质钙动力学或 AE6 的测量。
为了克服生理条件下实时AE研究的一些关键局限性，并能够研究钙动力学和AE之间的相互作用，生成了一个独特的小鼠模型。在该小鼠模型中，由基因编码的 Ca2+ 传感器 （GCaMP3） 和 mCherry 组成的融合蛋白被表达并使用 acrosin 启动子和信号转导肽2 靶向顶体。靶向双GCaMP3-mCherry传感器能够使用显微镜和单细胞兴奋剂递送系统，在生理条件下同时实时测量活精子中的钙浓度和顶体内容物状态（图1）。作为顶体基质的一个组成部分，膜融合和 AE 将导致精子中光稳定和 pH 不敏感的 mCherry 荧光丢失，因为这种蛋白质从顶体囊泡中扩散出来。在这方面，该模型反映AE的时间和发生的能力类似于顶体靶向GFP小鼠系7,8,9的益处。
该转基因小鼠品系中使用的 GCaMP3 变体的 KD 约为 400 μM，Ca2+ 的动态范围为 10-4-10-3 M10，适用于该囊泡。我们发现，GCaMP3 的这种特征组合可以揭示质膜和顶体外膜 （OAM） 之间的融合孔形成2。融合孔检测是由于孔径太小而无法使 AcroSensE 蛋白从顶体中分散出来（通过顶体内容物丢失），同时提供膜“通道”，使 Ca2+ 离子能够流入顶体腔，导致 GCaMP3 的荧光强度增加。
明亮的、单体的、非钙敏感的荧光蛋白mCherry支持顶体的可视化，而GCaMP3信号微弱（例如，在Ca2+ 结合之前， 图2），重要的是，它还允许识别适合成像的顶体完整精子细胞。
以下协议描述了独特的AcroSensE小鼠模型的利用以及实验中使用的显微镜方法，以高空间和时间分辨率研究AE和精子钙动力学。

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		<tr>
			<td>100x oil objective&#160;</td>
			<td>Olympus Japan</td>
			<td>UPlanApo,</td>
			<td></td>
		</tr>
		<tr>
			<td>2-hydroxypropyl-b-cyclodextrin&#160;</td>
			<td>Sigma</td>
			<td>C0926</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm coverslip dish, 1.5 thickness</td>
			<td>MatTek Corp.&#160;</td>
			<td>P35G-1.5-20-C&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round-bottomed tube</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capilarries</td>
			<td>Sutter Instrument Co. CA USA</td>
			<td>B200-156-10</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus Japan</td>
			<td>Olympus FluoView&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated tip&#160;</td>
			<td>TipOne, USA Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H7006</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ&#160;</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/plugins/index.html</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid</td>
			<td>Sigma</td>
			<td>G5889</td>
			<td></td>
		</tr>
		<tr>
			<td>Live-Cell Microscope Incubation Systems&#160;</td>
			<td>TOKAI HIT Shizuoka, Japan</td>
			<td>Model STX</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller&#160;</td>
			<td>Sutter Instrument Co. CA USA</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic transfer pipette&#160;</td>
			<td>FisherBrand&#160;</td>
			<td>13-711-6M</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine&#160;</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvic acid</td>
			<td>Sigma</td>
			<td>107360</td>
			<td></td>
		</tr>
		<tr>
			<td>Single cell delivery system</td>
			<td>Parker, Hauppauge, NY</td>
			<td>Picospritzer III</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time imaging of acrosomal calcium dynamics and exocytosis in live mouse sperm

顶体胞吐作用 （AE） 是精子的单个胞吐囊泡与质膜融合，是一个复杂的、依赖钙的过程，对受精至关重要。然而，我们对钙信号转导如何调节 AE 的理解仍然不完整。特别是，顶体内钙动力学与导致 AE 的中间步骤之间的相互作用尚不明确。在这里，我们描述了一种方法，该方法提供了对顶体钙动力学及其与膜融合和随后顶体囊泡胞吐作用的关系的空间和时间见解。该方法利用一种新型转基因小鼠，表达顶体靶向胞吐传感器（AcroSensE）。该传感器结合了与 mCherry 融合的基因编码钙指示剂 （GCaMP）。这种融合蛋白专门设计用于同时观察顶体钙动力学和膜融合事件。实时监测活体 AcroSensE 精子中的顶体钙动力学和 AE 是使用高帧率成像和可靶向单个精子的兴奋剂递送系统实现的。该协议还提供了几个用于量化和分析原始数据的基本方法示例。由于 AcroSensE 模型是基因编码的，因此可以通过使用现成的遗传工具（例如与其他小鼠遗传模型杂交或基于基因编辑 （CRISPR） 的方法）来增强其科学意义。通过这种策略，可以解决其他信号通路在精子获能和受精中的作用。总之，这里描述的方法提供了一种方便有效的工具来研究特定亚细胞区室（精子顶体）中的钙动力学，以及这些动力学如何调节导致膜融合和顶体胞吐作用的中间步骤。

图2 提供了一个简化的图示，显示了成功刺激精子后预期的荧光变化的顺序。 图 2 的上图显示了 GCaMP3 荧光强度的变化，其中信号最初变暗（基线顶体钙浓度低于 GCaMP3 KD），当钙离子 通过 融合孔进入时，荧光亮度增加。最后，在 AE 时，由于扩散和传感器向细胞外空间的损失而丢失信号。 图 2 的底板显示了 mCherry 荧光强度...

Watch this Scientific Journal Video about A Live Cell Imaging Technique to Study Calcium Signaling and Acrosome Exocytosis in Mouse Sperm at JoVE.com

作者聚焦：一种研究小鼠精子中钙信号传导和顶体胞吐作用的活细胞成像技术 

这里描述的AcroSensE小鼠模型和活细胞成像方法为研究精子顶体亚细胞区室中的钙动力学以及它们如何调节导致膜融合和顶体胞吐作用的中间步骤提供了一种新方法。

活小鼠精子顶体钙动力学和胞吐作用的实时成像

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...

我们的研究目标是更好地了解调节精子功能的分子机制，包括获能和受精等过程。具体来说，我们正在探索膜脂质如何激活钙信号传导和顶体胞吐作用。该协议解决了我们对获能，精子激活，钙信号传导和顶体胞吐作用之间相互作用的理解差距。该协议的优点包括在生理条件下对精子进行成像，以及它能够同时监测钙信号传导和顶体胞吐作用，而无需复杂的预加载或站立程序。

real-time-imaging-acrosomal-calcium-dynamics-exocytosis-live-mouse

Research

JoVE Journal

Developmental Biology

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

911 次观看.  Cornell University. 我们的研究目标是更好地了解调节精子功能的分子机制，包括获能和受精等过程。具体来说，我们正在探索膜脂质如何激活钙信号传导和顶体胞吐作用。该协议解决了我们对获能，精子激活，钙信号传导和顶体胞吐作用之间相互作用的理解差距。该协议的优点包括在生理条件下对精子进行成像，以及它能够同时监测钙信号传导和顶体胞吐作用，而无需复杂的预加载或站?...

视频: 作者聚焦：一种研究小鼠精子中钙信号传导和顶体胞吐作用的活细胞成像技术 

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

体细胞重编程的动力学测量和实时可视化

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

科研

发育生物学

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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 ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

人肠道 Organoids 上皮屏障通透性的实时测量

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...