Name: proplatelet formation dynamics of mouse fresh bone marrow explants
Uploaded: 2021-05-20T15:00:00
Description: Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

作者希望感谢Jean-Yves Rinckel，Julie Boscher，Patricia Laeuffer，Monique Freund，Ketty Knez-Hippert的技术援助。这项工作得到了ANR（国家研究机构）Grant ANR-17-CE14-0001-01和ANR-18-CE14-0037的支持。

所有动物实验均按照欧洲标准2010/63 / EU和斯特拉斯堡大学动物实验伦理委员会（斯特拉斯堡动物实验管理局）进行。
1. 试剂的制备
<ol><li>按 表1所述制备试剂。<ol>
<li>对于库存I，分别溶解每种粉末。确保制剂的渗透压高于295 mOsm/L。该溶液可以在4°C下储存一年。</li>
		<li>对于Tyrode的缓冲液制备，按照 表1所述制作溶液，用蒸馏水调节体积至100mL，并?...

<ol>
	<li>Thiery, J. P., Bessis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+platelet+genesis;+in+vitro+study+by+cinemicrophotography.">Mechanism of platelet genesis; in vitro study by cinemicrophotography.</a> Reviews in Hematology. 11 (2), 162-174 (1956).</li><li>Becker, R. P., De Bruyn, P. 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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hirudin+and+heparin+enable+efficient+megakaryocyte+differentiation+of+mouse+bone+marrow+progenitors.">Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors.</a> Experimental Cell Research. 318 (1), 25-32 (2012).</li><li>Aguilar, A., 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=Importance+of+environmental+stiffness+for+megakaryocyte+differentiation+and+proplatelet+formation.">Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.</a> Blood. 128 (16), 2022-2032 (2016).</li><li>Scandola, 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=Use+of+electron+microscopy+to+study+megakaryocytes.">Use of electron microscopy to study megakaryocytes.</a> Platelets. 31 (5), 589-598 (2020).</li><li>Eckly, A., 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=Characterization+of+megakaryocyte+development+in+the+native+bone+marrow+environment.">Characterization of megakaryocyte development in the native bone marrow environment.</a> Methods in Molecular Biology. 788, 175-192 (2012).</li><li>Eckly, A., 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=Abnormal+megakaryocyte+morphology+and+proplatelet+formation+in+mice+with+megakaryocyte-restricted+MYH9+inactivation.">Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation.</a> Blood. 113 (14), 3182-3189 (2009).</li><li>Eckly, A., 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=Proplatelet+formation+deficit+and+megakaryocyte+death+contribute+to+thrombocytopenia+in+Myh9+knockout+mice.">Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice.</a> Journal of Thrombosis and Haemostasis. 8 (10), 2243-2251 (2010).</li><li>Ortiz-Rivero, 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=C3G,+through+its+GEF+activity,+induces+megakaryocytic+differentiation+and+proplatelet+formation.">C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation.</a> Cell Communication and Signaling. 16 (1), 101 (2018).</li><li>Junt, T., 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317 (5845), 1767-1770 (2007).</li><li>Zhang, Y., 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=Mouse+models+of+MYH9-related+disease:+mutations+in+nonmuscle+myosin+II-A.">Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A.</a> Blood. 119 (1), 238-250 (2012).</li><li>Malara, A., 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=Extracellular+matrix+structure+and+nano-mechanics+determine+megakaryocyte+function.">Extracellular matrix structure and nano-mechanics determine megakaryocyte function.</a> Blood. 118 (16), 4449-4453 (2011).</li><li>Balduini, A., 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=Adhesive+receptors,+extracellular+proteins+and+myosin+IIA+orchestrate+proplatelet+formation+by+human+megakaryocytes.">Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes.</a> Journal of Thrombosis and Haemostasis. 6 (11), 1900-1907 (2008).</li></ol>

在这里，我们描述了一种简单且低成本的体外方法，以评估巨核细胞延长在骨髓中生长的丙酸盐的效率。小鼠骨髓外植体模型有四个主要优点。首先，不需要高级技术技能。其次，获得巨核细胞延伸丙酸盐所需的时间相当短，外植体方法仅为6小时，而从小鼠祖细胞开始的常规培养方法至少为4天。第三，鉴于只需要少量的组织并且获得的结果是可重复的，它将所需的小鼠数量减少到最低限?...

骨髓外植体技术最初由Thiéry和Bessis于1956年开发，用于将大鼠巨核细胞细胞质延伸的形成描述为血小板形成的初始事件1。使用相差和电影摄影技术，这些作者表征了成熟的圆形巨核细胞转化为"鱿鱼状"血小板细胞，其细胞质延伸显示出伸长和收缩的动态运动。这些手臂逐渐变薄，直到它们变成丝状，沿着手臂和尖端有小肿胀。这些典型的巨核细胞伸长，在体外和液体介质中获得，与固定骨髓中观察到的血小板有一定的相似性，其中巨核细胞通过正弦壁突出长延伸进入血液循环2，3。1994年TPO的发现和克隆，允许在培养物中区分巨核细胞，能够形成类似于骨髓外植体4，5，6中描述的丙酸盐延伸。然而，巨核细胞成熟在培养条件下的效率要低得多，特别是骨髓成熟的巨核细胞的广泛内部膜网络在培养的巨核细胞中发育不足，阻碍了对血小板生物发生机制的研究7，8。
我们在这里详细介绍了基于Thierry和Bessis的骨髓外植体模型，以实时跟踪小鼠巨核细胞的丙酸盐形成，这些巨核细胞在其原生环境中已经完全成熟，从而避免了可能的 体外 伪影和误解。在野生型成年小鼠中获得的结果被提出来说明巨核细胞扩展丙酸盐的能力，它们的形态和丙酸盐的复杂性。我们还引入了一种快速量化策略进行质量验证，以确保巨核细胞记录过程中的数据准确性和稳健性。这里介绍的协议是该方法的最新版本，作为书籍章节9发布。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 mL syringes</td>
			<td>Terumo</td>
			<td>SS+05S1</td>
			<td></td>
		</tr>
		<tr>
			<td>21-gauge needles</td>
			<td>BD Microlance</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2.6H2O</td>
			<td>Sigma</td>
			<td>21108</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverwall Incubation Chambers</td>
			<td>Electron Microscopy Sciences</td>
			<td>70324-02</td>
			<td>Depth : 0,2 mm</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H-3375</td>
			<td>pH adjusted to 7.5</td>
		</tr>
		<tr>
			<td>Human serum albumin</td>
			<td>VIALEBEX</td>
			<td>authorized medication : n&#176; 3400956446995</td>
			<td>20% (200mg/mL -100mL)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma</td>
			<td>BVBW8448</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glass</td>
			<td>Electron Microscopy Sciences</td>
			<td>72200-40</td>
			<td>22 mm x 55 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica Microsystems SA, Westlar, Germany</td>
			<td>DMI8 - 514341</td>
			<td>air lens</td>
		</tr>
		<tr>
			<td>microscope camera</td>
			<td>Leica Microsystems SA, Westlar, Germany</td>
			<td>K5 CMS GmbH -14401137</td>
			<td>image resolution : 4.2 megapixel</td>
		</tr>
		<tr>
			<td>Mouse serum</td>
			<td>BioWest</td>
			<td>S2160-010</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4.H2O</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>PSG 100x</td>
			<td>Gibco, Life Technologies</td>
			<td>1037-016</td>
			<td>10,000 units/mL penicillin, 10,000 &#956;g/mL streptomycin and 29.2 mg/mL glutamine</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose D (+)</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
	</tbody>
</table>

proplatelet formation dynamics of mouse fresh bone marrow explants

巨核生成术的最后阶段导致成熟巨核细胞的细胞质延伸，即所谓的丙酸盐。关于使用 体外分化的巨核细胞形成丙酸盐的很多知识;然而，越来越多的证据表明，传统的培养系统并不能忠实地概括骨髓内部发生的分化/成熟过程。在这份手稿中，我们提出了一种最初由Thierry和Bessis于1956年描述的外植体方法，以可视化在其原生环境中成熟的巨核细胞，从而规避潜在的伪影和误解。通过冲洗小鼠的股骨收集新鲜的骨髓，切成0.5mm横截面，并置于含有生理缓冲液的37°C孵育室中。巨核细胞在外植体外围逐渐可见，并在与摄像机耦合的倒置显微镜下观察长达6小时。随着时间的推移，巨核细胞改变其形状，一些细胞具有球形，而另一些细胞则发展出厚的延伸或延伸许多具有广泛分支的薄丙酸盐。进行定性和定量调查。该方法具有简单，可重复和快速的优点，因为存在许多巨核细胞，并且通常其中一半在6小时内形成丙酸盐，而培养的小鼠巨核细胞为4天。除了对突变小鼠的研究外，该方法的一个有趣的应用是直接评估丙酸盐延伸过程中的药理学药物，而不会干扰培养物中可能发生的分化过程。

定性结果。 在实验开始时，所有细胞都压实在骨髓部分。细胞在外植体的外围需要30分钟才能变得清晰可见。然后可以通过它们的大尺寸识别巨核细胞，然后可以随着时间的推移（大小，形状，动态，丙酸盐延伸和血小板释放）研究它们的进化（图2A）。小巨核细胞的直径在20至30μm之间，其细胞核是多叶的，而成熟的圆形巨核细胞较大（直径>30μm），具有扩大?...

Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

在这里，我们详细介绍了骨髓外植体方法，从样品制备到显微载玻片分析，以评估在其生理环境中分化的巨核细胞形成丙酸盐的能力。

小鼠新鲜骨髓外植体的丙酸盐形成动力学

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...

该程序的总体目标是实时跟踪小鼠巨核细胞形成的丙酸盐，这些巨核细胞在其天然环境中成熟。这种方法具有简单，可重复和快速的优点。人们可以分析许多巨核细胞，并在短短六个小时内可视化丙酸盐的形成，而不是像体外小鼠巨核细胞那样等待培养的第一天。这种方法的一个有趣的应用是研究药物治疗或基因突变的效果的可能性，特别是在丙酸盐延伸的步骤中。这里没有对分化过程的干扰，如体外培养的情况。此视频有助于确保冲洗和切割骨髓的这些关键步骤。它还解释了如何对形态学和巨核细胞进行分类，这对于表征易栓性疾病中的缺陷非常有用。首先，将Tyrode的缓冲液加热到37摄氏度，然后打开显微镜的加热室，将温度调到37摄氏度。准备所有必要的工具，例如计时器，孵育室，5毫升21号注射器，镊子，剃须刀片，巴斯德移液器，载玻片和15毫升离心管。通过将21号针头引入膝盖侧的股骨开口，用两毫升Tyrode缓冲液冲洗骨髓，然后缓慢按压柱塞以取回完整的骨髓圆柱体。使用三分钟升塑料移液器小心轻轻地将完整的骨髓转移到载玻片上。切掉在冲洗时可能被压缩的骨髓末端。然后使用锋利的剃须刀片切割0.5毫米厚的横向薄切片，以避免损坏巨核细胞。使用塑料移液器，将10个切片收集到一个含有Tyrode缓冲液的一毫升管中。小心地将切片转移到直径为13毫米的孵育室。吸出缓冲液并将体积调节至30微升Tyrode缓冲液，并补充5%小鼠血清。将截面放置在一定距离的位置。用22×55毫米的盖滑盖密封自粘室，倾斜盖滑以避免形成气泡。将腔室置于37摄氏度的加热室中。启动天文台表，在37摄氏度下运行实验六个小时，制作视频记录巨核细胞的转化。30分钟后，骨髓细胞逐渐迁移到外植体的外围，形成单层。孵育一小时后，可以通过它们的大尺寸和多叶核来鉴定巨核细胞。巨核细胞的数量在孵育三小时后增加，有些具有较长的扩展。绘制地图以定位孵育室中的每个部分。一小时后，识别可见的巨核细胞，它们是每个部分外围的巨型多叶细胞，并在图纸上绘制它们的位置。在三小时和六小时后重复此过程。手动计数巨核细胞，并在孵育室密封后三小时和六小时根据其形态进行分类。它们分为小，大，有厚的意图，和丙酸盐延伸。在映射的帮助下，它们可以随着时间的推移而跟踪其演变。结果表示为在每个观察时间每个类的百分比。传统上，在野生型小鼠骨髓的外周可见的巨核细胞中有一半在六小时时延长丙酸盐。在圆形巨核细胞的命运之后，随着时间的推移捕获顺序图像，以观察它们如何形成丙烯酸盐。尝试此过程时要记住的一件重要事情是在实验期间将外植体保持在37摄氏度。不同的温度可以改变结果。有趣的是，外植体方案可以在内向显微镜实验后使用。最后，骨髓外植体中的巨核细胞将是天然荧光的，并且它们的行为可以很容易地被研究。这使得可以对相同的动物进行不同的处理，从而减少小鼠的数量。总之，外植体方法快速，简单，并且提供了许多关于天然巨核细胞对外部丙酸盐的能力的信息。由此产生的定性和定量发现是我们理解易栓性疾病的关键。在生理或病理背景下。

Research

JoVE Journal

Biology

Culture of myeloid dendritic cells from bone marrow precursors

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

科研

生物学

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

代骨髓衍生的双光子成像使用的小鼠树突状细胞

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to 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 ...

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

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

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) ...

Osteoclast Derivation from Mouse Bone Marrow

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 ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

大鼠骨髓破骨衣原体的分离、纯化及鉴别

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

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

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

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 ...

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

小鼠骨髓造血干细胞和祖细胞以及基质生态位细胞的流式细胞术分析

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...