Name: isolation and culture of resident cardiac macrophages from the murine sinoatrial and atrioventricular node
Uploaded: 2021-05-07T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node at JoVE.com

这项工作得到了中国国家留学基金委（CSC，致夏瑞）、德国心血管研究中心（DZHK;81X2600255 致 S. Clauss、81Z0600206 致 S. Kääb、81Z0600204 致 CS）、科罗纳基金会（S199/10079/2019 致 S. Clauss）、SFB 914（项目 Z01 致 S. Massberg）、ERA-NET on 心血管疾病（ERA-CVD;01KL1910 至 S. Clauss）和 Heinrich-and-Lotte-Mühlfenzl Stiftung（致 S. Clauss）的支持。资助者在手稿准备中没有任何作用。

动物护理和所有实验程序均按照慕尼黑大学动物护理和伦理委员会的指导方针进行，对小鼠进行的所有程序均获得德国慕尼黑巴伐利亚州政府的批准。C57BL6 / J小鼠被商业获得。
1. 准备工作
<ol><li>制备细胞分选缓冲液（表1）并储存在4°C。 注意：在整个实验过程中，细胞分选缓冲液应始终在冰上。</li>
	<li>在消化前不久准备消化缓冲液（表1），因?...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationship+in+the+sinus+and+atrioventricular+nodes.">Structure-function relationship in the sinus and atrioventricular nodes.</a> Pediatric Cardiology. 33 (6), 890-899 (2012).</li><li>Clauss, 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=Animal+models+of+arrhythmia:+classic+electrophysiology+to+genetically+modified+large+animals.">Animal models of arrhythmia: classic electrophysiology to genetically modified large animals.</a> Nature Review Cardiology. 16 (8), 457-475 (2019).</li><li>Hulsmans, M., 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=Macrophages+Facilitate+Electrical+Conduction+in+the+Heart.">Macrophages Facilitate Electrical Conduction in the Heart.</a> Cell. 169 (3), 510-522 (2017).</li><li>Rosas, M., 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+transcription+factor+Gata6+links+tissue+macrophage+phenotype+and+proliferative+renewal.">The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal.</a> Science. 344 (6184), 645-648 (2014).</li><li>Schulz, 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=A+lineage+of+myeloid+cells+independent+of+Myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of Myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>Epelman, 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=Embryonic+and+adult-derived+resident+cardiac+macrophages+are+maintained+through+distinct+mechanisms+at+steady+state+and+during+inflammation.">Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.</a> Immunity. 40 (1), 91-104 (2014).</li><li>Gordon, S., Pluddemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+macrophages:+heterogeneity+and+functions.">Tissue macrophages: heterogeneity and functions.</a> BMC Biology. 15 (1), 53 (2017).</li><li>Bajpai, G., 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+human+heart+contains+distinct+macrophage+subsets+with+divergent+origins+and+functions.">The human heart contains distinct macrophage subsets with divergent origins and functions.</a> Nature Medicine. 24 (8), 1234-1245 (2018).</li><li>Davies, L. C., Jenkins, S. J., Allen, J. E., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages.">Tissue-resident macrophages.</a> Nature Immunology. 14 (10), 986-995 (2013).</li><li>Xia, R., 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=Whole-mount+immunofluorescence+staining,+confocal+imaging+and+3D+reconstruction+of+the+sinoatrial+and+atrioventricular+node+in+the+mouse.">Whole-mount immunofluorescence staining, confocal imaging and 3D reconstruction of the sinoatrial and atrioventricular node in the mouse.</a> Journal of Visualized Experiments. (166), e62058 (2020).</li><li>van Weerd, J. H., Christoffels, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+and+function+of+the+cardiac+conduction+system.">The formation and function of the cardiac conduction system.</a> Development. 143 (2), 197-210 (2016).</li><li>Ye Sheng, X., 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=Isolation+and+characterization+of+atrioventricular+nodal+cells+from+neonate+rabbit+heart.">Isolation and characterization of atrioventricular nodal cells from neonate rabbit heart.</a> Circulation: Arrhythmia and Electrophysiology. 4 (6), 936-946 (2011).</li><li>Murray, P. J., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+and+pathogenic+functions+of+macrophage+subsets.">Protective and pathogenic functions of macrophage subsets.</a> Nature Reviews Immunology. 11 (11), 723-737 (2011).</li><li>Pinto, A. R., 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=Revisiting+cardiac+cellular+composition.">Revisiting cardiac cellular composition.</a> Circulation Research. 118 (3), 400-409 (2016).</li><li>Bajpai, G., 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=Tissue+resident+CCR2-+and+CCR2++cardiac+macrophages+differentially+orchestrate+monocyte+recruitment+and+fate+specification+following+myocardial+injury.">Tissue resident CCR2- and CCR2+ cardiac macrophages differentially orchestrate monocyte recruitment and fate specification following myocardial injury.</a> Circulation Research. 124 (2), 263-278 (2019).</li><li>Honold, L., Nahrendorf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+and+monocyte-derived+macrophages+in+cardiovascular+disease.">Resident and monocyte-derived macrophages in cardiovascular disease.</a> Circulation Research. 122 (1), 113-127 (2018).</li><li>Leid, J., 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=Primitive+embryonic+macrophages+are+required+for+coronary+development+and+maturation.">Primitive embryonic macrophages are required for coronary development and maturation.</a> Circulation Research. 118 (10), 1498-1511 (2016).</li></ol>

在这篇手稿中，我们描述了一种方案，用于以高纯度富集来自SAN和AVN区域的心脏常驻巨噬细胞。
巨噬细胞根据其解剖位置和功能表型分为亚群。它们还可以响应可变的微环境信号从一种功能表型切换到另一种功能表型13。与骨髓和肝脏等其他器官相比，心脏组织包含较低百分比的免疫细胞和较低的每个细胞亚群的绝对数量14。因此，细胞分?...

An erratum was issued for: <a href="https://www.jove.com/t/62236">Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node</a>. The Authors section was updated from:
Ruibing Xia123 
Simone Loy1 
Stefan K&#228;&#228;b13 
Anna Titova1 
Christian Schulz123 
Steffen Massberg123 
Sebastian Clauss123 
1University Hospital Munich, Department of Medicine I Ludwig-Maximilian-Unversity Munich (LMU) 
2Walter Brendel Center of Experimental Medicine (WBex) LMU Munich 
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance (MHA)
to:
Ruibing Xia123 
Simone Loy1 
Stefan K&#228;&#228;b13 
Anna Titova1 
Christian Schulz123 
Steffen Massberg123 
Sebastian Clauss123 
1University Hospital Munich, Department of Medicine I Ludwig-Maximilian-University Munich (LMU) 
2Insitute of Surgical Research at the Walter Brendel Center of Experimental Medicine, University Hospital Munich, Ludwig-Maximilians-University (LMU) 
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance (MHA)

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62236">Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node</a>. The Authors section was updated.

Erratum: Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

窦房结 （SAN） 在生理上启动电脉冲，因此是心脏的主要起搏器。房室结 （AVN） 将电脉冲从心房传导到心室，并充当辅助起搏器1。一般来说，电脉冲的产生和传导是一个复杂的过程，可以通过各种因素2进行调节，包括SAN/AVN区域中的常驻巨噬细胞。Hulsmans等人最近的一项研究表明，心脏驻留巨噬细胞的特定群体富含AVN，并作为保持稳定心跳的关键参与者3。他们发现巨噬细胞与心肌细胞电偶联，可以改变偶联心肌细胞的电特性。作者还指出，这种与巨噬细胞交错的传导细胞也存在于心脏传导系统的其他组件中，例如SAN。
目前，尚不完全清楚常驻心脏巨噬细胞的表型是否在心脏区域之间有所不同。然而，已经表明，组织微环境可以影响组织巨噬细胞的转录和增殖更新4。此外，由于心肌细胞表型已被证明在区域之间是不同的，因此巨噬细胞对心肌细胞的功能作用也可能是区域特异性的，即使巨噬细胞表型本身可能相同。因此，需要对特定的心脏区域进行进一步的研究。
最近的研究表明，在稳定状态下，组织驻留巨噬细胞在产前建立，独立于确定性造血而产生，并持续到成年期5。然而，在巨噬细胞耗竭后或心脏炎症期间，Ly6chi单核细胞有助于补充心脏巨噬细胞群6。涉及遗传谱系追踪、共生、命运映射和细胞追踪的研究表明，器官和组织中存在多种组织驻留巨噬细胞群，以及可能与其个体发生相关的巨噬细胞亚群的不同细胞行为7，8，9。
常驻心脏巨噬细胞的表征得益于磁活化细胞分选（MACS）和荧光活化细胞分选的使用。这些方法对于通过用细胞表面标记物标记从多个组织组分中分离特定细胞群特别有用。这不仅导致分离的免疫细胞类型的纯度更高，而且还允许表型分析。在这里，我们提出了一个方案，包括磁珠包被的细胞，然后进行荧光活化细胞分选，以富集从SAN和AVN区域特异性分离的心脏驻留巨噬细胞。
为了探究心脏驻留巨噬细胞在传导系统中的特性及其对心脏传导和心律失常发生的功能，SAN和AVN的精确定位和解剖至关重要。对于SAN和AVN的显微解剖，解剖标志用于区域识别10。简而言之，SAN位于上腔静脉和右心房的交界处。AVN位于Koch三角形内，其前边界为三尖瓣的隔叶，后部与Todaro11的肌腱接壤。我们还提供小鼠SAN和AVN的准确显微切割程序，并通过组织学和免疫荧光染色得到证实。
分离的常驻巨噬细胞可用于进一步的实验，例如RNA测序，或者可以回收和培养两周以上，允许各种体外实验。因此，我们的协议描述了免疫节律学家非常有价值的程序。 表 1 显示了所需所有解决方案的组成，图 1 显示了 SAN 和 AVN 的显微切割特征。 图 2 是 SAN 和 AVN 本地化的示意图。 图3 显示了SAN和AVN的组织学染色（Masson的三色和免疫荧光染色）。 图4 显示了通过荧光激活细胞分选分离心脏驻留巨噬细胞的分步门控策略。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer system</td>
			<td>Hugo Sachs Elektronik</td>
			<td>34-0458, 34-1030, 73-4911, 34-0415, 73-4910</td>
			<td>Includes an induction chamber, a gas evacuation unit and charcoal filters</td>
		</tr>
		<tr>
			<td>Modified Bain circuit</td>
			<td>Hugo Sachs Elektronik</td>
			<td>73-4860</td>
			<td>Includes an anesthesia mask for mice</td>
		</tr>
		<tr>
			<td>Surgical Platform</td>
			<td>Kent scientific</td>
			<td>SURGI-M</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">In vivo instrumentation</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>91500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue forceps</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue pins</td>
			<td>Fine Science Tools</td>
			<td>26007-01</td>
			<td>Could use 27G needles as a substitute</td>
		</tr>
		<tr>
			<td colspan="4">General lab instruments</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Sunlab</td>
			<td>D-8040</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette,volume 10ul, 100ul, 1000ul</td>
			<td>Eppendorf</td>
			<td>Z683884-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RH basic</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Microscopes</td>
		</tr>
		<tr>
			<td>Dissection stereo- zoom microscope</td>
			<td>vwr</td>
			<td>10836-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica microscope</td>
			<td>Leica microsystems</td>
			<td>Leica DM6</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Flow cytometry machine</td>
		</tr>
		<tr>
			<td>Beckman Coulter</td>
			<td>Beckman coulter</td>
			<td>MoFlo Astrios</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Software</td>
		</tr>
		<tr>
			<td>FlowJo v10</td>
			<td>FlowJo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">General Lab Material</td>
		</tr>
		<tr>
			<td>0.2 &#181;m syringe filter</td>
			<td>sartorius</td>
			<td>17597</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>27G needle</td>
			<td>BD Microlance 3</td>
			<td>300635</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Polypropylene conical Tube</td>
			<td>FALCON</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Thermo scientific</td>
			<td>7632160</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes</td>
			<td>Eppendorf</td>
			<td>30121872</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Chemicals</td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Sigma</td>
			<td>20-158</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>100063</td>
			<td>Component of TEA</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biozym</td>
			<td>850070</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Worthington Biochemical</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase XI</td>
			<td>Sigma</td>
			<td>C7657</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (1X) Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma</td>
			<td>F2442-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin &#8722; Streptomycin</td>
			<td>Sigma</td>
			<td>P4083</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41966029</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Drugs</td>
		</tr>
		<tr>
			<td>Fentanyl 0.5&#160;mg/10&#160;ml</td>
			<td>Braun Melsungen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane 1 ml/ml</td>
			<td>Cp-pharma</td>
			<td>31303</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen 5L</td>
			<td>Linde</td>
			<td>2020175</td>
			<td>Includes a pressure regulator</td>
		</tr>
		<tr>
			<td colspan="4">Antibodies</td>
		</tr>
		<tr>
			<td>Anti-mouse Ly6C FITC (clone HK1.4)</td>
			<td>BioLegend</td>
			<td>Cat# 128006</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Anti-mouse F4/80 PE/Cy7(clone BM8)</td>
			<td>BioLegend</td>
			<td>Cat# 123114</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Anti-mouse CD64 APC (clone X54-5/7.1)</td>
			<td>BioLegend</td>
			<td>Cat# 139306</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b APC/Cy7(clone M1/70)</td>
			<td>BioLegend</td>
			<td>Cat# 101226</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 PE (clone 30-F11)</td>
			<td>BioLegend</td>
			<td>Cat# 103106</td>
			<td>diluted to 1:100</td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate (DAPI)</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>diluted to 1:1000</td>
		</tr>
		<tr>
			<td colspan="4">Animals</td>
		</tr>
		<tr>
			<td>Mouse, C57BL/6</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of resident cardiac macrophages from the murine sinoatrial and atrioventricular node

常驻心脏巨噬细胞已被证明可以促进心脏中的电传导。生理性心律由窦房结 （SAN） 中产生的电脉冲启动，然后通过房室结 （AVN） 传导至心室。为了进一步研究常驻巨噬细胞在心脏传导系统中的作用，有必要从SAN和AVN中适当分离常驻巨噬细胞，但这仍然具有挑战性。在这里，我们提供了一种方案，用于在小鼠心脏中可靠地显微解剖SAN和AVN，然后对常驻巨噬细胞进行分离和培养。
无论是位于终腔嵴与上腔静脉交界处的 SAN，还是位于科赫三角形顶点的 AVN，均被鉴定和显微解剖。通过对使用Masson三色染色和抗HCN4进行的组织学分析来确认正确的位置。
然后对显微解剖的组织进行酶消化以获得单细胞悬液，然后用针对细胞类型特定表面标志物的特定抗体组孵育。这允许通过荧光激活细胞分选来识别、计数或分离不同的细胞群。为了将心脏驻留巨噬细胞与心肌中的其他免疫细胞（尤其是募集的单核细胞来源的巨噬细胞）区分开来，需要一种精心设计的门控策略。首先，检测淋巴谱系细胞并将其排除在进一步分析之外。然后，通过CD45和CD11b的高表达以及Ly6C的低表达来鉴定髓系细胞和常驻巨噬细胞。通过细胞分选，可以在体外培养分离的心脏巨噬细胞数天以进行进一步研究。因此，我们描述了一种分离位于心脏传导系统内的心脏驻留巨噬细胞的方案。我们讨论了显微解剖和消化SAN和AVN的陷阱，并提供了一种门控策略，通过荧光激活细胞分选可靠地鉴定，计数和分选心脏巨噬细胞。

我们描述了一种从SAN和AVN区域分离心脏驻留巨噬细胞的实用程序。为了确认正确的解剖，进行了Masson的三色染色和免疫荧光HCN4染色（图3）12。通过这个协议，我们可以从一个完整的心脏收集大约60，000个巨噬细胞。图4显示了用于分选心脏巨噬细胞的门控策略。活的常驻心脏巨噬细胞被鉴定为CD45 + CD11b + F4 / 80 + CD64 ...

Watch this Scientific Journal Video about Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node at JoVE.com

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

这里介绍的方案提供了一种从小鼠心脏的窦房结（SAN）和房室结（AVN）区域分离心脏驻留巨噬细胞的分步方法。

小鼠窦房和房室结常驻心脏巨噬细胞的分离和培养

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

我们描述了一种通过荧光激活细胞分选特异性从小鼠的鼻窦和AV结中分离心脏驻留微噬细胞的显微解剖和随后分离的方案。通过对鼻窦和V结进行精确的显微切割，然后结合磁性和荧光激活细胞分选，我们能够特异性地从窦和V淋巴结中分离出高纯度的常驻巨噬细胞。从心脏不同区域鉴定和分离心脏驻留巨噬细胞可以进一步研究它们的表型及其对心脏电生理学的区域特异性影响。分离心脏后，在解剖显微镜下用冰冷的一次性PBS在解剖皿中进行显微解剖程序。使用心脏解剖标志物（如主动脉、肺动脉、冠状窦和左心室或右心室）来确定心脏的左右和前后。确定方向后，将心脏与前部放在培养皿底部，以露出后大静脉。沿着心室和心房之间的凹槽切割以去除大部分心室。保留心房部分。通过将销钉插入左心室自由壁和LAA的其余部分，将心脏固定在解剖盘上。通过三尖瓣和上腔静脉向上切开剩余的无右心室壁。将 RA 和 RV 翻转开并固定 RAA 以暴露 RA 腔腔间区域和房间隔。对于 SAN 的显微解剖，沿着平行于终嵴的房间隔切开心脏以分离腔间区域以获得 SAN 样本。将样品放入冰上的空1.5毫升微量离心管中。对于 AVN 的显微解剖，将心脏的其余部分穿过房间隔和室间隔相邻的组织，朝上房间隔的右侧。查看心内膜表面的右心房，寻找科赫三角形，该三角形将向前与三尖瓣隔叶的铰链线接壤，后部与托达罗的肌腱接壤。在底部观察到冠状窦的孔口。用手术刀切碎 SAN 和 AVN 组织。然后向每个样品中加入500微升新鲜制备的消化缓冲液，并从微量离心管壁上洗涤所有切碎的组织。轻轻移液以帮助消化样品并在涡旋机上均质组织。消化后，通过将组织悬浮液通过40微米细胞过滤器，将其转移到新鲜的15毫升离心管中。用额外的五毫升细胞分选缓冲液冲洗细胞过滤器以停止消化。除去上清液。用90微升细胞分选缓冲液重悬细胞沉淀后，在15毫升离心管中的细胞悬液中每1000万个细胞中加入10微升CD45微珠。将样品充分混合。在用微珠和抗体混合物孵育细胞悬液期间，通过将磁柱连接到合适的磁选器上，然后将收集管置于磁柱下方来制备磁分离装置。通过在色谱柱顶部添加500微升细胞分选缓冲液冲洗并让缓冲液通过来制备磁性色谱柱。在细胞分选缓冲液通过时，立即将细胞悬液涂在色谱柱上，避免气泡。用500微升细胞分选缓冲液洗涤色谱柱三次。在色谱柱上加入一毫升细胞分选缓冲液。从磁选机中取出色谱柱并将其放在新的收集管上。通过牢固地应用柱塞立即冲洗色谱柱，以获得含有磁性标记细胞的流通液。应用未染色的样品和补偿管后，调整每个通道的电压，使正负峰对齐到轴的适当位置。使用 DAPI 作为可行性标记，按照文本手稿中所述设置门控策略。检查流式细胞术图表，确认感兴趣的细胞群正确显示在图表上。根据后续实验的需要将分选的细胞收集到培养基或缓冲液中。为了确认正确的解剖，使用免疫荧光和Masson的三色染色对SAN和AVN进行了鉴定。此处显示了SAN和AVN中HCN4阳性传导系统细胞的免疫荧光染色，以及SAN和AVN的Masson三色染色。用于常驻心脏巨噬细胞细胞分选的门控策略有助于通过排除y轴与x轴前向散射区域中的双峰来识别单核细胞。DAPI排除了死细胞。对CD45阳性白细胞进行活细胞门控，然后对CD11b阳性髓细胞进行门控。通过F4/80和CD64的表达来鉴定心脏巨噬细胞，然后通过淋巴细胞抗原六表达进行分层。在明场显微镜下观察新鲜分选的细胞。在荧光团PE通道下观察时，细胞CD45呈阳性。当在荧光团APC大小为七通道下观察时，分选细胞的相同视图显示CD11b阳性细胞。使用荧光团PE尺寸七通道鉴定F4/80阳性表型。三重阳性细胞被鉴定为心脏驻留巨噬细胞。在培养基中培养长达六天的分离心脏巨噬细胞用白色箭头表示，而黑色箭头表示死细胞。分选的巨噬细胞可用于进一步的功能研究，如膜片钳实验或表达研究，如RNA测序或蛋白质组学研究。

Research

JoVE Journal

Medicine

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

可视化<em&gt;肺炎链球菌</em&gt;在心脏Microlesions和随后的心脏重构

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation and Purification of Murine Cardiac Pericytes

鼠心围细胞的分离与纯化

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

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 ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

穆林·斯特拉特·甘利翁的位置、解剖和分析

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...