Name: bone marrow-derived macrophage production
Uploaded: 2013-11-22T14:30:00
Description: Watch this Scientific Journal Video about Bone Marrow-derived Macrophage Production at JoVE.com

这项工作是由法国国家科学研究中心（PICS 2012-2014 EG）和由REGIONE坎帕尼亚（LR N.5，2002年3月28日至乔凡娜莫托拉）的资助。菲利波·孔蒂是科学合作基金会&#39;&#39; Infectiopole南德意志集团的资深会员。&#39;&#39;尼古拉Boucherit是法国教育部研究和技术的资深会员。资金来源有在研究设计，数据收集，数据分析，发布的决定，或手稿的准备没有任何作用。

操守准则该协议为动物处理批准了我们的机构动物伦理委员会“行政法院科学研究都中心去形成等德RECHERCHE实验医学 - Chirurgical”（CFREMC，项目许可证10-300122013埃里克GHIGO）从埃克斯 - 马赛大学符合规则的DécretN°的1987年10月19日87-848。该实验是在Faculté德MEDECI​​NE德拉蒂莫（实验许可证号13.385埃里克GHIGO）执行。 1。物质与文化媒体准备<ol><li>消毒2钳子，?...

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	<li>Rutherford, M. S., Witsell, A., Schook, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+generating+functionally+heterogeneous+macrophages:+chaos+revisited.">Mechanisms generating functionally heterogeneous macrophages: chaos revisited.</a> J. Leukocyte Biol. 53, 602-618 (1993).</li><li>Van Furth, R., Gallin, J. I., Goldstein, I. M., Snyderman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inflammation: Basic Principles and Clinical Correlates. 112, 325-336 (1992).</li><li>Adams, D., Halmiton, T., Gallin, J. I., Goldstein, I. M., Snyderman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inflammation: Basic Principles and Clinical Correlates. 112, 325-336 (1992).</li><li>Morris, L., Graham, C. F., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+in+haemopoietic+and+other+tissues+of+the+developing+mouse+detected+by+the+monoclonal+antibody+F4/80.">Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80.</a> Development. 112, 517-526 (1991).</li><li>Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phagosome:+compartment+with+a+license+to+kill.">The phagosome: compartment with a license to kill.</a> Traffic. 8, 311-330 (2007).</li><li>Underhill, D. M., Ozinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+microbes:+complexity+in+action.">Phagocytosis of microbes: complexity in action.</a> Annu. Rev. Immunol. 20, 825-852 (2002).</li><li>Castagna, A., Polati, R., Bossi, A. M., Girelli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte/macrophage+proteomics:+recent+findings+and+biomedical+applications.">Monocyte/macrophage proteomics: recent findings and biomedical applications.</a> Expert Rev. Proteomics. 9, 201-215 (2012).</li><li>Benoit, M., Desnues, B., Mege, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+in+bacterial+infections.">Macrophage polarization in bacterial infections.</a> J. Immunol. 181, 3733-3739 (2008).</li><li>Barry, A. O., 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=Impaired+stimulation+of+p38alpha-MAPK/Vps41-HOPS+by+LPS+from+pathogenic+Coxiella+burnetii+prevents+trafficking+to+microbicidal+phagolysosomes.">Impaired stimulation of p38alpha-MAPK/Vps41-HOPS by LPS from pathogenic Coxiella burnetii prevents trafficking to microbicidal phagolysosomes.</a> Cell Host Microbe. 12, 751-763 (2012).</li><li>Henry, R. M., Hoppe, A. D., Joshi, N., Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uniformity+of+phagosome+maturation+in+macrophages.">The uniformity of phagosome maturation in macrophages.</a> J. Cell Biol. 164, 185-194 (2004).</li><li>Sundaramurthy, V., 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=Integration+of+Chemical+and+RNAi+Multiparametric+Profiles+Identifies+Triggers+of+Intracellular+Mycobacterial+Killing.">Integration of Chemical and RNAi Multiparametric Profiles Identifies Triggers of Intracellular Mycobacterial Killing.</a> Cell Host Microbe. 13, 129-142 (2013).</li><li>Boltz-Nitulescu, 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=Differentiation+of+rat+bone+marrow+cells+into+macrophages+under+the+influence+of+mouse+L929+cell+supernatant.">Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant.</a> J. Leukocyte Biol. 41, 83-91 (1987).</li></ol>

本文所述的协议细节的方法来产生大量的BMDMs的。 BMDM是原代细胞和有生物功能的巨噬细胞和单核细胞分化的性能，因为有成熟，而相比之下，巨噬细胞的细胞系，这是不成熟。 BMDMs可用于遗传筛查（RNAi）技术，药物筛选，功能研究，宿主 - 病原体相互作用的研究和调查等诸多领域。 本文中所呈现的过程是很简单的，并且不需要任何特殊的设备，因此，它可以很容易地在实验...

巨噬细胞功能的显著方面是其在先天免疫和适应性免疫的作用。因为它们的吞噬惰性颗粒，细菌或寄生虫的能力，巨噬细胞是抵御外来入侵者的第一道防线。一旦内化，微生物被吞噬溶酶体中降解。巨噬细胞也送招募信号和呈递抗原给其他的免疫细胞，如T淋巴细胞。巨噬细胞衍生自单核细胞。单核细胞出现在骨髓从髓样干细胞和迁移到外周血和各种组织中，其中它们分化成巨噬细胞。据估计，一个健康的成年小鼠包含分布在整个身体中各种器官和组织（ 表1）1,2，大约10 8巨噬细胞。因为他们来适应自己的微环境3,4能力的巨噬细胞显示大表型和功能的多样性。最重要的macrop哈格财产是他们的抗菌活性，这是由它们的能力定义吞噬微生物并摧毁他们。吞噬反应是由是由​​微生物接触刺激复杂信号网络的激活所定义，因此，巨噬细胞响应各种刺激适当地调节基因表达。吞噬后，微生物在一个叫做吞噬溶酶体结构消除了，但是，许多病原微生物开发战略，颠覆巨噬细胞5的杀菌功能。由不同的微生物物种所利用的颠覆机制的多样性是证明了吞噬6过程的复杂性和吞噬溶酶体的生物合成。传染病是人类主要的健康问题，以及众多的机制和分子参与巨噬细胞的抗菌活性。此外，是由微生物劫持的杀菌性能的目标依然是未知，因此，有一个电子xplosion中的宿主 - 病原体相互作用的进化，遗传和生化方面已经就续约巨噬细胞科学的关注兴趣。目前，大部分的研究领域是做对巨噬细胞的细胞系，其中从初级巨噬细胞的吞噬活性，细胞因子产生和氧化爆发的调控有所不同。此外，它们更适合于显微镜。为了研究巨噬细胞的相互作用，其病原体是推荐使用初级巨噬细胞，如骨髓源性巨噬细胞（BMDMs），它表现出多种生理功能。此外，它可以工作在转基因BMDMs，因为这些巨噬细胞可能直接从转基因小鼠中分离出来，并与新颖的技术，如慢病毒转染的可用性，它们的基因表达图谱可以通过基因过表达或RNA干扰而改变。在这里，我们描述了一个程序来区分小鼠骨髓米箭头为巨噬细胞，将提供大量的巨噬细胞在7天为各种功能分析，如7蛋白质组学，转录8，细胞内运输研究9，动态研究10，遗传筛选（RNAi）技术和药物筛选11。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco Life Technologies</td>
			<td>21969-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Gibco Life Technologies</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco Life Technologies</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco Life Technologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Lonza</td>
			<td>BEM515F</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#956;m Nylon</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#956;m Nylon</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (100/20 mm)</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td>culture treated</td>
		</tr>
		<tr>
			<td>Petri dishes (35/10 mm)</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

bone marrow-derived macrophage production

巨噬细胞是先天免疫和适应性免疫应答的重要组成部分，它们是抵御外来入侵者，因为他们强大的杀菌活动的第一线。巨噬细胞广泛分布在整个身体和存在于淋巴器官，肝，肺，胃肠道，中枢神经系统，骨和皮肤。因为他们的再分配，他们参与广泛的生理和病理过程。巨噬细胞是高度通用的细胞，能够识别微环境的改变和维持组织稳态。许多病原体已经进化机制，使用巨噬细胞作为木马的生存，在复制和感染人类和动物的整个身体进行传播。在宿主 - 病原体相互作用的进化，遗传和生化方面的兴趣最近的爆炸已经更新有关的巨噬细胞科学的关注。在这里，我们描述了一个程序从小鼠骨髓，将提供大量的巨噬细胞为研究宿主 - 病原体相互作用，以及其他工艺分离和培养的巨噬细胞。

该方法的目的是为了容易地得到大量的巨噬细胞中几天。骨髓细胞的制备示于图1。从后腿骨头收集砸在研钵。一旦定居巨噬细胞是从骨髓细胞去除制剂，骨髓细胞与GM-CSF（第0天）。 3天后，细胞，它呈圆形和非贴壁培养前，开始分化成巨噬细胞和坚持（ 图1A）。流式细胞术分析使用F4/80和HLA-DR的标记显示，细胞表达两种标记从而证明它们分化 ​​成巨噬细胞（ 图1B...

Watch this Scientific Journal Video about Bone Marrow-derived Macrophage Production at JoVE.com

Bone Marrow-derived Macrophage Production

巨噬细胞一直被认为是先天免疫和适应性免疫应答的重要组成部分。知识的最近的爆炸关于巨噬细胞和微生物之间的相互作用的演化，遗传和生化方面已经更新的科学关注的巨噬细胞。本文介绍了一种方法，从小鼠骨髓巨噬细胞分化。

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Research

JoVE Journal

Immunology and Infection

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

弓形虫囊肿壁形成激活骨髓衍生的巨噬细胞和Bradyzoite条件

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

科研

免疫与感染

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

偏光M1和M2骨髓来源的巨噬细胞的使用实时外流量分析代谢表征

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

鼠后肢长骨解剖和骨髓隔离

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

抗体药物对小鼠骨髓及腹腔巨噬细胞活化作用的评价

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...