Name: preparation of a non-cardiomyocyte cell suspension for single-cell rna sequencing from a post-myocardial infarction adult mouse heart
Uploaded: 2023-02-03T12:40:00
Description: Watch this Scientific Journal Video about Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart at JoVE.com

这项工作得到了浙江省自然科学基金（LQ22H020010）的支持。

所有实验均按照浙江大学实验动物护理和使用指南进行，并经浙江大学动物咨询委员会批准。
1.左前降冠状动脉结扎术（LAD结扎术）手术
注意：使用八周龄雄性C57BL / 6J小鼠作为模型。心肌梗死后2周摘取心脏。 如前所述进行左前降冠状动脉结扎手术11。
<ol><li>手术前用70%乙醇对手术器械进行消毒。</li>
	<li>称量小鼠，然后?...

<ol>
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本文旨在描述一种在心肌梗死后从小鼠心脏中分离单个非心肌细胞的方案。该方案可用于高质量分离后MI微环境中不同类型的细胞，包括免疫细胞，内皮细胞和成纤维细胞。三个基本因素对于获得用于单细胞测序的高质量细胞悬液至关重要。第一个是酶消化的设置。重要的是控制消化时间以及消化酶的体积和浓度，以确保细胞活力和分离效率。在这种情况下，我们适当增加酶的浓度并延长消化时?...

心肌梗塞（MI）是最常见的心血管疾病之一，其死亡率在全世界都在上升1。心肌梗死是由周围心肌的血液供应不足引起的，这可能是动脉粥样硬化斑块破裂时冠状动脉阻塞的结果。尽管经皮冠状动脉介入治疗 （PCI） 降低了急性心肌梗死患者的死亡率，但心肌梗死后心力衰竭的高患病率仍然是一个问题2。心肌梗死后心力衰竭的关键病理生理学是身体对心脏损伤的代偿反应，包括用非收缩性纤维化瘢痕替换死去的心肌。这些适应性反应在很大程度上依赖于由多种细胞类型和心脏组织细胞之间的相互作用介导的局部炎症，这种炎症现在被认为是减少纤维化疤痕形成的潜在治疗靶点，从而防止心肌梗死后心力衰竭3，4。有趣的是，梗死部位的微环境在MI1，5的不同阶段经历了浸润细胞类型和功能的时间依赖性转变。许多研究表明，非心肌细胞（例如免疫细胞、成纤维细胞和内皮细胞）在心肌梗死后炎症和组织修复中起核心作用5，6。近年来，单细胞测序已被广泛用作阐明非心肌细胞在后MI微环境中的参与和功能的有力工具7，8。 这为心肌梗死后损伤和修复的病理生理学以及针对心肌梗死后心力衰竭的潜在疗法的开发提供了见解。
高通量RNA测序（RNA-Seq）是一种使用下一代测序（NGS）7，8，9详细研究整个转录组的技术。最近，scRNA-Seq的发展彻底改变了生物医学研究领域。与传统的批量测序相比，scRNA-Seq在单细胞水平上分析基因表达谱和转录异质性7，8。该技术通过识别后MI微环境中的不同循环细胞类型并揭示心肌细胞和非心肌细胞之间的相互作用，显着促进了MI9，10细胞病理生理学的研究。这些发现进一步有助于发现心肌梗死后心力衰竭的新治疗靶点。一般来说，基于scRNA-seq的MI后实验包括三个主要部分：（1）MI后动物模型的建立;（2）细胞悬液的制备;（3）样本测序和数据分析。值得注意的是，制备细胞悬液是制备scRNA-Seq实验中最关键的一步，因为细胞悬液的质量决定了结果的准确性。
该协议旨在从MI后的心脏组织中提取非心肌细胞悬液;值得注意的是，包括维持细胞活力和分辨率的具体细节。同时，该方案中使用的设备，例如小鼠手术套件，啮齿动物呼吸机和离心机，可以在大多数动物实验中心和生物医学实验室中找到，因此，该方案的实验成本相对较低。此外，如果将梗死的时间点和部位视为变量，则该协议可用于模拟广泛的临床场景，特别是对于心肌梗死后并发症。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acridine Orange / Propidium Iodide Stain</td>
			<td>Logos biosystems</td>
			<td>F23001</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated&#160;Cell&#160;Counter</td>
			<td>Logos biosystems</td>
			<td>L20001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Servicebio</td>
			<td>G5001</td>
			<td>Cytoprotective effect</td>
		</tr>
		<tr>
			<td>Cell Counting Slides</td>
			<td>Logos biosystems</td>
			<td>L12001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td>Digestive enzymes</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma</td>
			<td>D4693</td>
			<td>Digestive enzymes</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td>Prevent cell clumping</td>
		</tr>
		<tr>
			<td>Falcon&#160;40&#956;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td>Remove cell clumps</td>
		</tr>
		<tr>
			<td>Falcon&#160;70&#956;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td>Remove undigested tissue and clumps</td>
		</tr>
		<tr>
			<td>Flow Cell Sorter</td>
			<td>Beckman Coulter</td>
			<td>B25982</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodophor</td>
			<td>OU QING SI</td>
			<td>10054963976859</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>FST</td>
			<td>12061-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic Forceps</td>
			<td>RWD</td>
			<td>F14012-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic Scissors</td>
			<td>RWD</td>
			<td>S11036-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline&#160;</td>
			<td>Servicebio</td>
			<td>G4202-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC Lysis Buffer</td>
			<td>Beyotime</td>
			<td>C3702-120ml</td>
			<td>Remove red blood cells</td>
		</tr>
		<tr>
			<td>Rib Retractor</td>
			<td>FST</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Ventilator</td>
			<td>Harvard</td>
			<td>730043</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>Solvent&#160;solution of enzyme</td>
		</tr>
		<tr>
			<td>Sterile Scissor</td>
			<td>RWD</td>
			<td>S14014-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of a non-cardiomyocyte cell suspension for single-cell rna sequencing from a post-myocardial infarction adult mouse heart

心肌梗塞（MI）是最常见的心血管疾病之一，全球死亡率都在上升。非心肌细胞占心脏细胞总数的一半以上，它们有助于心肌损伤时的适应性补偿，包括炎症反应、组织修复和瘢痕形成。为了研究MI后的心脏微环境，单细胞RNA测序（scRNA-seq）被广泛用于识别不同的心脏细胞类型和细胞间通讯。在scRNA-seq样品制备程序中，制备细胞悬液是最关键的步骤之一，因为细胞活力会影响scRNA-seq结果的质量。因此，我们设计了一种实验方案，用于从MI后小鼠心脏制备非心肌细胞悬液，并特别关注通过选择温和的消化酶，控制消化时间和应用荧光激活细胞分选（FACS）来提高细胞活力。最后，我们从通过该协议获得的非心肌细胞悬液中分离CD45 + 细胞，然后我们进行了scRNA-seq。

通过实施该协议的第2节获得了具有高活力的单细胞悬液。然而，仍然可以观察到细胞碎片（图1A）;因此，进行了荧光激活细胞分选（FACS）以进一步提高质量16。FACS后，平均细胞大小从9.6μm减小到9.1μm（ 表1），这表明FACS可以有效降低细胞悬液中细胞片段的比例 （图1B）。用于FACS的门控策略如图 <strong class="xf...

Watch this Scientific Journal Video about Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart at JoVE.com

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

在这里，我们描述了一种从心肌梗死后（MI）小鼠心脏中分离出足够数量的具有高活力的单个非心肌细胞的方案。这可用于随后的单细胞测序、流式细胞术分析和原代细胞培养。

制备用于心肌梗死后成年小鼠心脏单细胞RNA测序的非心肌细胞悬液

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

我们的方案以相对较低的成本确保单细胞悬液的细胞活力和分离效率。相对较低的成本和对实验室设备的要求是该技术的主要优点。单细胞测序揭示了非心肌细胞在心脏病理生理学中的异质性，也为心脏病的基础和临床研究提供了思路。该协议可用于对心肌梗死后心血管环境的研究，例如免疫细胞的参与。对于首次制备，我建议应特别注意样品胃蛋白酶和消化时间。一旦按照手稿中的描述制备缓冲液和溶液，将两周前发生心肌梗塞的安乐死小鼠仰卧在手术台上。用胶带固定它的后肢。要切除心脏，请用70%乙醇喷洒身体。在避免肝脏的同时，通过腹部皮肤和肌肉进行垂直切口。在不刺穿心脏的情况下，小心地打开胸部，用预冷的PBS溶液大量注入左心室，直到灌注液无色。使用镊子轻轻提起心脏，并用眼科剪刀切掉附着在其外部的多余组织。将心脏置于1.5毫升微量离心管中的一毫升预冷PBS中。将心脏转移到放置在冰上的24孔板的孔中。将150微升心脏解离缓冲液移入孔中，并用剪刀将心脏切成一毫米大小的碎片。将切碎的心脏组织转移到含有5毫升组织解离缓冲液的15毫升离心管中。将管子放入恒温往复摇床中。每 20 分钟上下移液悬浮液 10 次。为了去除团块和未消化的组织，使用25毫升细胞洗涤缓冲液通过70微米细胞过滤器过滤细胞悬液。冲洗离心管和细胞过滤器。接下来，通过40微米的细胞过滤器过滤细胞悬液以去除更多的细胞团块。将过滤后的细胞悬液在 300 x g 下在 4 摄氏度下离心五分钟。将细胞样品重悬于五体积的RBC裂解缓冲液中，并在室温下孵育5分钟。现在，如前所述，在细胞悬液和离心机中加入四体积的细胞洗涤缓冲液。弃去上清液后，将细胞重悬于10毫升细胞洗涤缓冲液中，并再次离心。在离心结束时，弃去上清液并将沉淀重悬于一毫升细胞洗涤缓冲液中。取18微升悬浮液在另一个离心管中，加入两微升吖啶橙碘化丙啶染色溶液。将10微升该染色溶液添加到计数室载玻片中。使用自动细胞计数器评估细胞数量和活力。在单细胞心脏悬液中观察到细胞碎片。荧光活化细胞分选有效地降低了细胞片段的比例和细胞聚集，从而减小了平均细胞大小。RNA测序证明了七种类型的免疫细胞的明显分离，如无监督聚类和减少的T分布随机邻居嵌入所证明的那样。每个免疫细胞都显示出经典标志物的高表达。心脏组织需要切得足够小，以避免消化不完全，这会导致细胞悬液中出现大量细胞块。通过该方案获得的单细胞悬液可以进一步应用于原代细胞培养物的流动分析。该协议有利于对心肌感染微环境的研究，这可能有助于研究人员超越心脏组织，将心肌感染视为一个系统性问题。

Research

JoVE Journal

Medicine

Myocardial Infarction and Functional Outcome Assessment in Pigs

心肌梗死和功能预后评估的猪

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

科研

医学

A Modified Method for Heterotopic Mouse Heart Transplantion

对于异位小鼠心脏移植技术的一种改进方法

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

An Isolated Working Heart System for Large Animal Models

一个孤立的工作心脏系统的大型动物模型

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

左冠状动脉前降支的小鼠结扎：对心肌梗死后重塑和心衰模型

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

临床样品RNA病毒的无偏深度测序

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Isolation of Atrial Myocytes from Adult Mice

从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

研究小鼠心肌梗死的低温损伤模型

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

建立干细胞治疗心肌梗塞心力衰竭的猪模型

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in G&#246;ttingen Minipigs and Landrace Pigs

Post-Myocardial Infarction Heart Failure in Closed-chest Coronary Occlusion/Reperfusion Model in GÃƒÆ’Ã‚Â¶ttingen Minipigs and Landrace Pigs

封闭胸冠状动脉闭塞/输液模型中的心肌梗塞后心力衰竭

The development of heart failure is the most powerful predictor of long-term mortality in patients surviving acute myocardial infarction (MI). There is an ...