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).
1. Material and Culture Media Preparation
<ol>
	<li>Sterilize two forceps, two scissors, two surgical blades, a mortar and a pestle.</li>
	<li>Obtain complete DMEM containing 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin and 100 &#956;g/ml streptomycin .</li>
	<li>Dilute 10x PBS in sterile distillated water to obtain 1x PBS.</li>
	<li>Obtain ice-cold 1x PBS, ice-cold complete DMEM, complete DMEM warmed to 37 &#176;C.</li>
</ol>
2. Preparation of L929 Cell Supernatants
<ol>
	<li>Grow L929 cells to confluence (twenty 175 cm2 flasks) in complete DMEM at 37 &#176;C, 5% CO2.</li>
</ol>
Note: Granulocyte-macrophage colony stimulating factor (GM-CSF) is required to induce hematopoietic cell differentiation into macrophages12. L929 cells produce GM-CSF.
<ol start="2">
	<li>At confluence, replace culture media with fresh complete DMEM. Transfer flasks to 32 &#176;C, 5% CO2 for 10 days.</li>
	<li>Collect, pool and centrifuge the supernatants at 750 x g for 10 min. Discard cell pellets.</li>
	<li>Store supernatants in 15 ml tubes and store at -20 &#176;C.</li>
</ol>
3. Bone Marrow-derived Macrophage (BMDM) Preparation
<ol>
	<li>Sacrifice 1 mouse by cervical dislocation.
	<ol>
		<li>Use sterile surgical blades throughout the experiment. Disinfect the skin with 70% alcohol. Make an incision at the top of each hind leg and pull the skin down towards the foot to expose the muscle.</li>
		<li>Cut off the hind legs, remove the skin with sterile scissors and sterile forceps. Place legs within a sterile Petri dish (35/10 mm) containing sterile, ice-cold 1x PBS (5 ml).</li>
		<li>Remove the flesh and muscles that are adhering to the bones with sterile scissors and forceps.</li>
	</ol>
	</li>
	<li>Transfer the bones into a new, sterile Petri dish (35/10 mm) containing ice-cold, sterile 1x PBS (5 ml). Wash the bones two times with 5 ml ice-cold, sterile 1x PBS.</li>
	<li>Transfer the bone into a sterile mortar containing 5 ml ice-cold, sterile 1x PBS.</li>
	<li>Cut the tibia from the femur at the joint with sterile scissors. Smash the bones gently in a sterile mortar containing 5 ml ice-cold, sterile 1x PBS using a pestle.</li>
	<li>Collect the supernatant in ice-cold 15&#160;ml tubes. Repeat this step 3x.</li>
	<li>Filter through a 70 &#956;m Nylon cell strainer to remove solid fragments. Centrifuge the filtrate at 450 x g for 10 min at 4 &#176;C.</li>
	<li>Gently discard the supernatant. Dissociate the pellet in 10 ml red blood cell lysis buffer for 30 sec. Add 20 ml ice-cold, complete DMEM.</li>
</ol>
Note: The aim of this step is to remove contaminating red blood cells; thus, this step must be performed within 2 min to avoid hematopoietic cell alteration by the red blood cell lysis buffer.
<ol start="8">
	<li>Centrifuge at 450 x g for 10 min at 4 &#176;C. Gently discard the supernatant. Dissociate the pellet in 20 ml complete DMEM that has been warmed to 37 &#176;C.</li>
	<li>Transfer the dissociated cells into 2 Petri dishes (100/20 mm). Incubate them for 4 hr at 37 &#176;C.</li>
	<li>Collect the supernatants in 50 ml tubes at room temperature. Discard the dishes that contain the resident macrophages.</li>
</ol>
Note: The objective of this step is to eliminate the resident bone marrow macrophages by their ability to adhere to culture-treated plastic. These resident macrophages may be used in other experiments.
<ol start="11">
	<li>Centrifuge the collected supernatants at 450 x g for 10 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Dissociate gently the pellet in 10 ml complete DMEM containing 15% L929 cell supernatant. Filter the cells through a 40 &#956;m Nylon cell strainer.</li>
	<li>Recover the filtrate. Add the collected filtrate (10 ml) to 140 ml complete DMEM that has been supplemented with 15% L929 cell media.</li>
	<li>Distribute 10 ml of the cell suspension per Petri dish (15 Petri dishes, 100/20 mm). Incubate the cells at 37 &#176;C, 5% CO2.</li>
	<li>Grow cells for 3 days</li>
	<li>Add 10 ml complete DMEM that has been supplemented with 15% L929. Incubate cells for 4 additional days.</li>
</ol>
Note: Monitor cell growth periodically with an inverted microscope (steps 3.14-3.16). Adherent macrophages will be observed after 3 days of culture.
4. Harvesting BMDMs
<ol>
	<li>Remove the supernatant. Wash BMDMs 2 times with complete DMEM</li>
	<li>Add 5 ml complete DMEM that has been warmed to 37 &#176;C. Detach BMDMs by gently scraping with a rubber policeman.</li>
	<li>Collect BMDMs in 50 ml tubes and centrifuge at 450 x g for 10 min. Gently dissociate cell pellets in 20 ml complete DMEM.</li>
	<li>Count BMDMs in the presence of trypan blue (no more than 10% mortality should be observed).</li>
</ol>
Note: Generally, approximately 6-7.5 x 107 macrophages are obtained from 15 Petri dishes (100/20 mm) of macrophages. There are approximately 4-5 x 106 macrophages/Petri dish (100/20 mm).
<ol start="5">
	<li>Prepare BMDMs as required for the experiment. After 16 hr in complete media at 37 &#176;C, 5% CO2, macrophages will again adhere to the support.</li>
</ol>
5. Storing BMDMs
<ol>
	<li>Collect isolated BMDMs (step 4.3). Centrifuge at 450 x g for 10 min. Note: BMDMs may be frozen in liquid nitrogen.</li>
	<li>Resuspend cell pellets in freezing media consisting of 10% DMSO and 90% FCS at a final concentration of 4 x 106 cells/ml and pipette 1 ml into each ampoule.</li>
	<li>Freeze the cells at a cooling rate of 1 &#176;C/min. After 24 hr, transfer the ampoule to a liquid nitrogen container for long-term storage.</li>
</ol>

<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>

There are no declared conflicts of interest.

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><tbody><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 &amp;mu;m Nylon</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>Cell strainer 40 &amp;mu;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 ...

bone-marrow-derived-macrophage-production

Research

JoVE Journal

Immunology and Infection

73.0K Views.  Aix-Marseille Université. 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.

Video: Bone Marrow-derived Macrophage Production

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 stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

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

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

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 M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

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

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

Author Spotlight: Developing a Reproducible Method for Isolating Bone Marrow-Derived Macrophages from Neonatal Mice to Advance Early Life Immune Responses

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

Bone Marrow-derived Macrophage Production

Summary

Explore More Videos

Chapters in this video

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

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

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages