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

This work was supported by the CNRS (PICS 2012-2014 to E.G.) and by a grant from Regione Campania (L.R. n.5, 28.03.2002 to Giovanna Mottola). Filippo Conti is a fellow of the Scientific Cooperation Foundation &#39;&#39;Infectiopole Sud.&#39;&#39; Nicola Boucherit is a fellow of the French Ministry for Research and Technology. The funding sources had no role in the study design, data collection, data analysis, decision to publish, or manuscript preparation.

Ethics Statement
The protocol for animal handling was approved by our institutional Animal Ethics Committee &#34;Conseil Scientifique du Centre de Formation et de Recherche Experimental M&#233;dico-Chirurgical&#34; (CFREMC, Project permit 10-300122013 to Eric Ghigo) from Aix-Marseille University in accord with the rules of D&#233;cret N&#176; 87-848 of 10/19/1987. The experiments were performed at the Facult&#233; de M&#233;decine de la Timone (Experimentation permit number 13.385 to Eric Ghigo)....

<ol>
	<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>

The protocol described herein details a method to produce large numbers of BMDMs. BMDM are primary cells and have the biological function and properties of macrophages differentiated from monocytes because there are mature, in contrast to macrophage cell lines, which are immature. BMDMs may be used for genetic screening (RNAi), drug screening, functional studies, host-pathogen interaction studies and many other areas of investigation.
The procedure presented herein is very simple and does not ...

A significant aspect of macrophage function is their role in innate and adaptive immunity. Because of their capacity to phagocytose inert particles, bacteria or parasites, macrophages are a first line of defense against foreign invaders. Once internalized, microbes are degraded within phagolysosomes. Macrophages also send recruitment signals for and present antigens to other immune cells such as T lymphocytes. Macrophages are derived from monocytes. Monocytes arise in the bone marrow from myeloid stem cells and migrate to peripheral blood and various tissues where they differentiate into macrophages. It is estimated that a healthy adult mouse contains approximately 108 macrophages that are distributed throughout the body in various organs and tissues (Table 1)1,2. Macrophages display great phenotypic and functional diversity because of their ability to adapt to their microenvironment3,4. The most important macrophage property is their microbicidal activity, which is defined by their capacity to phagocytose microbes and destroy them. The phagocytic response is defined by the activation of complex signaling networks that are stimulated by microbial contact; thus, macrophages modulate gene expression appropriately in response to diverse stimuli. After phagocytosis, microbes are eliminated in a structure called the phagolysosome; however, many pathogenic microbes have developed strategies to subvert the microbicidal function of macrophages5. The diversity of subversion mechanisms that are utilized by different microbial species is a testament to the complexity of the phagocytic process6 and phagolysosome biogenesis. Infectious diseases are major human health problems, and numerous mechanisms and molecules participate in macrophage antimicrobial activities. Furthermore, the targets of the microbicidal properties that are hijacked by microbes remain unknown; thus, there is an explosion of interest in the evolutionary, genetic, and biochemical aspects of host-pathogen interactions that has renewed scientific attention regarding macrophages. Currently, the majority of the research in the field is done on macrophages cell lines, which differ from primary macrophages in the phagocytic activity, the cytokines production and the regulation of the oxidative burst. In addition, they are less suitable for microscopy. To investigate the interaction macrophages-pathogens it is recommended to use primary macrophages, such as Bone Marrow Derived Macrophages (BMDMs), which exhibit more physiological features. Moreover, it is possible to work on genetically modified BMDMs, because these macrophages might be isolated directly from transgenic mice and, with the availability of novel technologies such as lentiviral transfection, their gene expression profile can be altered by gene overexpression or RNA interference. Here, we describe a procedure to differentiate murine bone marrow into macrophages that will provide large numbers of macrophages in 7 days for various functional analyses such as proteomics7, transcriptomics8, intracellular trafficking studies9, dynamic studies10, genetic screens (RNAi) and drug screening11.

<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

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

The aim of this method was to easily obtain large numbers of macrophages in few days. The bone marrow cell preparation is illustrated in Figure 1. Bones from the hind leg were collected and smashed in a mortar. Once the resident macrophages were removed from the bone marrow cell preparation, bone marrow cells were incubated with GM-CSF (Day 0). After 3 days, the cells, which were round and nonadherent before culture, start to differentiate into macrophages and adhere (Figure 1A). Cytomet...

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

Bone Marrow-derived Macrophage Production

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

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

Research

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Immunology and Infection

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

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

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Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

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

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

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

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

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

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

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