This work was supported by grants from Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de Minas Gerais (FAPEMIG), through Rede de Pesquisa em Doen&#231;as Infecciosas Humanas e Animais do Estado de Minas Gerais (RED-00313-16) and Rede Mineira de Engenharia de Tecidos e Terapia Celular - REMETTEC (RED-00570-16), and the Brazilian National Council for Scientific and Technological Development (CNPq).

This protocol was carried out in accordance with The National Council for the Control of Animal Experimentation (Concea) and with the approval of the Ethics and Use of Animals Committee (CEUA). C57BL/6 mice were purchased from Biot&#233;rio Central of Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil. Personal protective equipment (PPE) such as laboratory coats, gloves, and eye protection should be used at all steps described in this protocol.
1. Preparation of fibroblast L-929 lineage supernatant as a source of M-CSF
<ol>
	<li>Defrost L-929 fibroblast lineage cells (American Type Culture Collection Certified Cell Line-ATCC CCL1) and start the culture process immediately to preserve the viability.</li>
	<li>In a laminar flow biosafety cabinet, using a 1,000 &#181;L pipette, transfer the L-929 fibroblast lineage cells from the vial to a 50 mL sterile centrifuge tube.</li>
	<li>Use a serological pipette to add 50 mL of sterile phosphate-buffered saline (PBS), free from calcium and magnesium.</li>
	<li>Centrifuge at 300 x g, 4 &#176;C for 10 min. Discard the supernatant.</li>
	<li>Resuspend the pellet in 20 mL of Dulbecco&#8242;s modified Eagle&#8242;s medium-F12 supplemented with 10% fetal bovine serum (FBS) and 1 mL of penicillin/streptomycin (P/S) (DMEM/F12-10) using a serological pipette.</li>
	<li>Transfer the resuspended cells to a T75 cell culture flask.</li>
	<li>Incubate in a 37 &#176;C, 5% CO2 incubator until full confluence. 
	NOTE: While manipulating the flask, be careful to avoid wetting the cover with solution, as this can be a source of contamination. To obtain a convenient amount of supernatant, it is necessary to replicate the cells after they cover the full surface of the cell culture flask. L-929 cells are inhibited by contact. Warm trypsin/ethylenediaminetetraacetic acid (EDTA) solution, DMEM/F12-10, and sterile PBS to 37 &#176;C to start the cell culture expansion, as described in the following steps.</li>
	<li>Once full confluence is reached, discard the supernatant from the cell culture flask, inside the laminar flow biosafety cabinet.</li>
	<li>Wash the cell culture flask with 20 mL of sterile PBS using a serological pipette and discard the solution.</li>
	<li>Add 10 mL of trypsin/EDTA (0.05% solution) to the cell culture flask containing the adhered cells.</li>
	<li>Transfer the cell culture flask from laminar flow to a 37&#176;C, 5% CO2 incubator and incubate for 3 min.</li>
	<li>Shake the cell culture flask to dissociate the cells from the surface of the culture flask and use a microscope to ensure that all the cells have been dissociated.</li>
	<li>Inactivate the trypsin/EDTA (0.05% solution) with 10 mL of DMEM/F12-10 medium.</li>
	<li>Transfer the solution using a serological pipette to a 50 mL sterile centrifuge tube.</li>
	<li>Centrifuge at 300 x g at 4 &#176;C for 10 min. Discard the supernatant.</li>
	<li>Resuspend the pellet in 10 mL of DMEM/F12-10 medium.</li>
	<li>Add 1 mL of the cell suspension to a T175 cell culture flask (1:10 culture).</li>
	<li>Repeat the procedure to obtain ten T175 cell culture flasks.</li>
	<li>Add 50 mL of DMEM/F12-10 to each cell culture flask.</li>
	<li>Incubate in a 37 &#176;C, 5% CO2 incubator until full confluence. 
	NOTE: These steps guarantee 500 mL of L-929 cell supernatant. On the day 5, after the cells have covered the T175 cell culture flask surface, the supernatant will be enriched for M-CSF and must be collected14.</li>
	<li>Remove the cell culture flask from the incubator on day 5 after contact inhibition.</li>
	<li>Transfer the medium to a 50 mL sterile centrifuge tube.</li>
	<li>Centrifuge at 3,200 x g, at 4 &#176;C for 10 min.</li>
	<li>Collect the supernatant enriched with M-CSF and transfer the solution to a 50 mL sterile centrifuge tube. Store in a freezer at -20 &#176;C. 
	&#8203;NOTE: Centrifugation is extremely important to remove cells and debris from the supernatants.</li>
	<li>L-929 supernatant is a convenient and inexpensive source of macrophage colony-stimulating factor (M-CSF) and typically contains 5 to 10 ng/mL of M-CSF8. These supernatants also contain trace amounts of other cytokines and growth factors, but they do not exert a profound influence on macrophage physiology15. However, purified M-CSF can be purchased and used as an alternative to L-929 supernatants at concentrations of 1 to 2 ng/mL8.</li>
</ol>
2. Removal of the femur and tibia
<ol>
	<li>Proceed with 8-week-old, C57BL-6 wild-type male mouse euthanasia by cervical dislocation after carbon dioxide asphyxiation, in accordance with normative resolution number 18 of the National Council for Animal Experimentation (Concea) and with approval of the Ethics and Use of Animals Committee (CEUA).</li>
	<li>Soak the mouse with 70% ethanol solution and use sterile scissors to make a 1 cm incision along the abdomen.</li>
	<li>Remove the skin until the muscle of the hind legs is fully exposed.</li>
	<li>Remove the hind legs at hip height, being careful not to break the femur and tibia.</li>
	<li>Put the hind legs in a conical centrifuge tube with 70% ethanol solution. 
	&#8203;NOTE: Always use pathogen-free mice. Be sure to collect both legs to achieve a greater number of cells. Step 2&#160;is performed in a non-sterile environment (benchtop). Hence, it is important to remove the femur and tibia carefully so they remain intact to avoid bacterial contamination. The remaining steps of this procedure are carried out in a laminar flow biosafety cabinet. The time of exposure of the legs to 70% ethanol solution should be limited to 10 min. If the time is longer, bone marrow progenitors can be affected.</li>
</ol>
3. Obtain bone marrow progenitors from the lumen of the tibia and femur
<ol>
	<li>Remove the legs from the 70% ethanol solution and transfer them to sterile PBS.</li>
	<li>Using forceps and disinfectant wipes to remove all muscle and fascia. The bone should be clean after this step.</li>
	<li>Use a sterile surgical scalpel blade to cut the bone epiphysis on both ends to expose the bone marrow.</li>
	<li>Fill a 20 mL syringe with 10 mL of sterile PBS supplemented with 2% penicillin/streptomycin solution and connect a 26 G needle.</li>
	<li>Hold the bone using anatomical dissection forceps with one hand, and use the other to insert the needle into the bone cavity. Be careful to not crush the bone with the forceps.</li>
	<li>Wash out the inside of the bone with the PBS and collect the bone marrow in a sterile conical centrifuge tube. After this step, the bone cavity should appear white.</li>
	<li>Centrifuge the collected cells for 10 min at 300 x g at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the cell pellet in 1 mL of DMEM/F12-10.</li>
	<li>Homogenize and add another 9 mL of DMEM/F12-10 medium to bring the cells up to a total of 10 mL.</li>
</ol>
4. Macrophage culture
<ol>
	<li>Add 1 mL of cell progenitors to each of the 100 mm x 20 mm round plastic Petri dishes (total of 10 dishes), spreading the cells across the plates with a pipette to obtain a uniform distribution. Do not use tissue-culture treated dishes.</li>
	<li>Add 9 mL of DMEM/F12-10 supplemented with 20% of the L-929 cell supernatant to each dish.</li>
	<li>Incubate in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>On the 3rd day, add 10 mL of DMEM/F12-10 medium supplemented with 20% of the supernatant of L-929 cells to each dish. In this way, the culture dishes now have a total of 20 mL of culture medium, sufficient for the growth of the cells until the end of their maturation. 
	NOTES: Tissue culture-treated dishes must not be used because macrophages, as natural adherent cells marked by strong interactions with surfaces, will not easily dissociate from the dish later. Usually, cell progenitors from one mouse (two femurs) can be seeded in 10 culture dishes. Cell progenitors will transform to macrophages, which can be used from incubation day 7 to 10. The maturation confirmation is identified by changes on the morphology of macrophages. Mature macrophages are adherent and show heterogeneous morphology, explained by the emission of pseudopodia in different directions. Examples of these mature cells are shown in the representative results.</li>
</ol>
5. Harvesting macrophages
<ol>
	<li>Discard the supernatants from all the culture dishes.</li>
	<li>Wash each culture dish with 10 mL of warm (37 &#176;C) sterile Mg2+ and Ca2+ free PBS.</li>
	<li>Discard the solution from each dish.</li>
	<li>Pre-warm non-enzymatic cell dissociation solution to 37 &#176;C and add 3 mL to each dish. Incubate for 10 min in a 37 &#176;C, 5% CO2 incubator. Use an inverted microscope to visualize if the macrophages are dissociating from the culture dishes. 
	NOTE: The use of non-tissue culture treated plastic plates should allow the easy dissociation of cells and avoid the need to scrape the cells off the plate.</li>
	<li>Wash the dishes using a serological pipette with the non-enzymatic cell dissociation solution over and over, performing circular movements to confirm all the macrophages are dissociated from the dish.</li>
	<li>Collect the solution of all the culture dishes together to a conical 50 mL centrifuge tube.</li>
	<li>Add 10 mL of sterile pre-warmed PBS to the empty culture dishes and proceed as in step 5.4. This procedure is a repetition of step 5.4 and intends to guarantee that all the macrophages are collected.</li>
	<li>Centrifuge at 300 x g, 4 &#176;C for 10 min.</li>
	<li>Discard the supernatant and resuspend the pellet with 1 mL of DMEM/F12-10. Homogenize slowly and carefully up and down using the pipette to avoid damaging the cells.</li>
	<li>Prepare an aliquot of 10 &#181;L of macrophage solution diluted with 10 &#181;L of Trypan Blue to count the cells using the hemocytometer. 
	NOTE: At day 7, each dish will produce approximately 2 x 106 macrophages. This means that one mouse can produce approximately 2 x 107 primary macrophages when harvested in ten plates. Since M-CSF only drives the maturation of macrophages, the procedure guarantees a population of cells that is essentially just macrophages and free from other contaminating leukocytes16.</li>
</ol>

<ol>
	<li>Gomez Perdiguero, E., 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+macrophages+originate+from+yolk-sac-derived+erythro-myeloid+progenitors.">Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.</a> Nature. 518 (7540), 547-551 (2015).</li><li>van Furth, R., Cohn, Z. 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+origin+and+kinetics+of+mononuclear+phagocytes.">The origin and kinetics of mononuclear phagocytes.</a> The Journal of Experimental Medicine. 128 (3), 415-435 (1968).</li><li>Wynn, T. A., Chawla, A., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+biology+in+development,+homeostasis+and+disease.">Macrophage biology in development, homeostasis and disease.</a> Nature. 496 (7446), 445-455 (2013).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+physiological+roles+of+inflammation.">Origin and physiological roles of inflammation.</a> Nature. 454 (7203), 428-435 (2008).</li><li>Shapouri-Moghaddam, A., 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=Macrophage+plasticity,+polarization,+and+function+in+health+and+disease.">Macrophage plasticity, polarization, and function in health and disease.</a> Journal of Cellular Physiology. 233 (9), 6425-6440 (2018).</li><li>Davies, J. Q., 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=Isolation+and+culture+of+murine+macrophages.">Isolation and culture of murine macrophages.</a> Methods in Molecular Biology. 290, 91-103 (2005).</li><li>Jin, X., Kruth, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+macrophage+colony-stimulating+factor+differentiated+human+monocyte-derived+macrophages.">Culture of macrophage colony-stimulating factor differentiated human monocyte-derived macrophages.</a> Journal of Visualized Experiments. (112), e54244 (2016).</li><li>Gon&#231;alves, R., Mosser, D. 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+isolation+and+characterization+of+murine+macrophages.">The isolation and characterization of murine macrophages.</a> Current Protocols in Immunology. 111 (1), 1-16 (2015).</li><li>Varol, C., Mildner, A., Jung, 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:+development+and+tissue+specialization.">Macrophages: development and tissue specialization.</a> Annual Review of Immunology. 33, 643-675 (2015).</li><li>Andr&#233;s, V., Pello, O. M., Silvestre-Roig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+proliferation+and+apoptosis+in+atherosclerosis.">Macrophage proliferation and apoptosis in atherosclerosis.</a> Current Opinion in Lipidology. 23 (5), 429-438 (2012).</li><li>Pineda-Torra, I., Gage, M., de Juan, A., Pello, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture,+and+polarization+of+murine+bone+marrow-derived+and+peritoneal+macrophages.">Isolation, culture, and polarization of murine bone marrow-derived and peritoneal macrophages.</a> Methods in Molecular Biology. 1339, 101-109 (2015).</li><li>Edwards, J. P., Zhang, X., Frauwirth, K. A., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+functional+characterization+of+three+activated+macrophage+populations.">Biochemical and functional characterization of three activated macrophage populations.</a> Journal of Leukocyte Biology. 80 (6), 1298-1307 (2006).</li><li>Hamczyk, M. R., Villa-Bellosta, R., Andr&#233;s, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+macrophage+phagocytosis+assay.">In vitro macrophage phagocytosis assay.</a> Methods in Molecular Biology. 1339, 235-246 (2015).</li><li>Hosoe, 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=Induction+of+tumoricidal+macrophages+from+bone+marrow+cells+of+normal+mice+or+mice+bearing+a+colony-stimulating-factor-producing+tumor.">Induction of tumoricidal macrophages from bone marrow cells of normal mice or mice bearing a colony-stimulating-factor-producing tumor.</a> Cancer Immunology, Immunotherapy. 28 (2), 116-122 (1989).</li><li>Rice, H. 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=rM-CSF+efficiently+replaces+L929+in+generating+mouse+and+rat+bone+marrow-derived+macrophages+for+in+vitro+functional+studies+of+immunity+to+intracellular+bacteria.">rM-CSF efficiently replaces L929 in generating mouse and rat bone marrow-derived macrophages for in vitro functional studies of immunity to intracellular bacteria.</a> Journal of Immunological Methods. 477, 112693 (2020).</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> Journal of Leukocyte Biology. 41 (1), 83-91 (1987).</li><li>Weischenfeldt, J., Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+macrophages+(BMM):+isolation+and+applications.">Bone marrow-derived macrophages (BMM): isolation and applications.</a> Cold Spring Harbor Protocols. 2008 (12), (2008).</li><li>Austin, P. E., Mcculloch, E. A., Till, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+factor+in+L-cell+conditioned+medium+capable+of+stimulating+colony+formation+by+mouse+marrow+cells+in+culture.">Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture.</a> Journal of Cellular Physiology. 77 (2), 121-134 (1971).</li><li>Stanley, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+colony-stimulating+factor+(CSF-1).">Macrophage colony-stimulating factor (CSF-1).</a> Encyclopedia of Immunology. 116 (1983), 1650-1654 (1998).</li><li>Bou Ghosn, E. E., 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=Two+physically,+functionally,+and+developmentally+distinct+peritoneal+macrophage+subsets.">Two physically, functionally, and developmentally distinct peritoneal macrophage subsets.</a> Proceedings of the National Academy of Sciences. 107 (6), 2568-2573 (2010).</li><li>Ray, A., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+peritoneal+cavity+cells.">Isolation of mouse peritoneal cavity cells.</a> Journal of Visualized Experiments. (35), e1488 (2010).</li></ol>

The authors declare that they have no conflicts of interest.

Producing a population of bone marrow-derived macrophages is a basic step in many cell biology experimental models, especially when it is important to achieve a homogeneous population of primary cells. As mentioned, cell progenitors can only transform into macrophages in the presence of M-CSF. L-929 cell supernatants can be used as the main source of M-CSF. Other than the cost, there is no problem using recombinant M-CSF itself8,17. There is some evidence that re...

Monocytes and macrophages are mononuclear phagocytes that can be derived from progenitors in the bone marrow. Recent studies have reported that macrophages also originate from yolk sac-derived erythro-myeloid progenitors1. Regardless of their derivation, these leukocytes have important effector functions in homeostasis and inflammation2,3. Monocytes are cells from peripheral blood that can further differentiate into macrophages in the tissue2,4, whereas macrophages are heterogeneous cells that exhibit phenotypes and functions regulated by the local exposure of growth factors and cytokines5. Since macrophages show such functional diversity, they have been studied in many disease models. Thus, the in vitro culture of macrophages has become an important tool for understanding their physiology and their role in different diseases. The bone marrow is an important source of progenitor cells, including macrophage progenitors, which can be isolated and multiplied, exponentially increasing the number of macrophages obtained. In addition, bone marrow-derived macrophages are especially important to avoid the effects generated by the tissue microenvironment, since macrophages change their phenotype in response to different stimuli in tissues6,7. Bone marrow progenitors transform into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF)8. Bone marrow-derived macrophages cannot be distinguished from monocyte-derived macrophages by biochemical markers in tissue. These cells represent a highly homogeneous population of primary cells, that in many other respects are comparable to peritoneal macrophages6,9.
Because of their heterogeneous set of cellular functions, macrophages have long been thoroughly studied by investigators. These cells can be used in different experimental models, including infectious and inflammatory diseases, as they feature in these processes10,11. They can also be useful to investigate macrophage polarization in response to various microenvironmental stimuli12,13. Thus, a simple and reliable protocol is provided here for the purpose of obtaining high numbers of primary macrophages from mouse bone marrow.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >20-cc syringe</td>
			<td >DESCARPACK</td>
			<td >SI100S4G</td>
			<td >Sterile syringe</td>
		</tr>
		<tr >
			<td >26-G needles</td>
			<td >BD</td>
			<td >497AQDKT7</td>
			<td >Sterile needles</td>
		</tr>
		<tr >
			<td >50-ml conical centrifuge tube</td>
			<td >SARSTEDT</td>
			<td >62547254</td>
			<td >Plastic conical tubes suitable for centrifugation</td>
		</tr>
		<tr >
			<td >70% ethanol solution</td>
			<td >EMFAL</td>
			<td >490</td>
			<td >Ethanol solution for esterilization</td>
		</tr>
		<tr >
			<td >Anatomical dissection forceps</td>
			<td >3B SCIENTIFIC</td>
			<td >W1670</td>
			<td >Maintain in sterile beaker containing 70% ethanol solution</td>
		</tr>
		<tr >
			<td >C57Bl/6 wild type mouse</td>
			<td >Purchased from Biot&#233;rio Central at Federal University of Minas Gerais</td>
			<td >Not applicable</td>
			<td >Mice must be specific-pathogen-free, age between 6 and 10 weeks. Mice need be accommodated at least one week earlier for recovering from the stress of transportation</td>
		</tr>
		<tr >
			<td >Cell culture flask T175</td>
			<td >GREINER</td>
			<td >C7481-50EA</td>
			<td >T-175 flask, canted neck, surface area 75 cm2, with filter cap, DNase free, RNase free</td>
		</tr>
		<tr >
			<td >Cell culture flask T75</td>
			<td >GREINER</td>
			<td >C7231-120EA</td>
			<td >T-75 flask, canted neck, surface area 75 cm2, with filter cap, DNase free, RNase free</td>
		</tr>
		<tr >
			<td >Disinfecting or baby Wipes&#8321;</td>
			<td >CLOROX</td>
			<td >Not applicable</td>
			<td >It helps cleaning the bone</td>
		</tr>
		<tr >
			<td >Distilled Water</td>
			<td >GIBCO</td>
			<td >15230</td>
			<td >Sterile distilled water</td>
		</tr>
		<tr >
			<td >DMEM/F12-10</td>
			<td >Not applicable</td>
			<td >Not applicable</td>
			<td >Add 10 mL of Fetal Bovine Serum (FBS) and 1 mL of Penicillin/ Streptomycin (P/S) to DMEM/F12 (q.s.p. 100 mL)</td>
		</tr>
		<tr >
			<td >DMEM/F12-10 + supernatant of L-929 cells</td>
			<td >Not applicable</td>
			<td >Not applicable</td>
			<td >Add 20% of supernatant of L929 cells culture on DMEM/F-12-10</td>
		</tr>
		<tr >
			<td >Dulbecco&#8242;s Modified Eagle&#8242;s Medium - F12 (DMEM/F12)</td>
			<td >GIBCO</td>
			<td >12500096</td>
			<td >DMEM: F-12 Medium contains 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM sodium pyruvate, and 1200 mg/L sodium bicarbonate.Resuspend powder to 1 liter of 
			distilled water and add 3,7 g of sodium bicarbonate. Adjust pH to 7,2 and filter with 0,22 &#181;M. Storage at 2 - 8 &#176;C freezer</td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum, certified, heat inactivated, United States (FBS)</td>
			<td >GIBCO</td>
			<td >10082147</td>
			<td >Enrichment for DMEM-F12</td>
		</tr>
		<tr >
			<td >Hemocytometer</td>
			<td >SIGMA-ALDRICH</td>
			<td >Z359629</td>
			<td >Used to count macrophages at microscopy</td>
		</tr>
		<tr >
			<td >L-929 cells</td>
			<td >SIGMA-ALDRICH</td>
			<td >ATCC # CCL-1</td>
			<td >L-929 is a lineage of mouse fibroblast cells used as a source of macrophage colony stimulating factor (M-CSF)</td>
		</tr>
		<tr >
			<td >Nikon TI eclipse&#160;</td>
			<td >NIKON&#160;</td>
			<td >Not applicable</td>
			<td >Nikon TI Eclipse is a fluorescence and phase contrast microscope</td>
		</tr>
		<tr >
			<td >Non-enzymatic cell dissociation solution</td>
			<td >CELLSTRIPER (CORNING)</td>
			<td >25-056-CI</td>
			<td >Non-enzimatic cell dissociation solution remove macrophages from the plate without damaging them</td>
		</tr>
		<tr >
			<td >Non-treated round culture dishes 100 &#215; 20&#8211;mm</td>
			<td >CORNING</td>
			<td >CLS430591</td>
			<td >Do not use tissue culture treated petri dishes or any tissue culture treated plate</td>
		</tr>
		<tr >
			<td >P3199 Penicillin G Potassium Salt</td>
			<td >USBIOLOGICAL</td>
			<td >113-98-4</td>
			<td >Antibiotics</td>
		</tr>
		<tr >
			<td >Penicillin and streptomycin (P/S) solution</td>
			<td ></td>
			<td ></td>
			<td >Use 0,26 grams of penicillin and 0,40 grams of streptomycin. Mix with 40 mL of sterile PBS. Inside a horizontal laminar flow cabinet, use a 0,22 &#181;M filter and store aliquots of 1.0 mL in 1.5 ml microfuge tubes in the freezer (-20&#176;C)</td>
		</tr>
		<tr >
			<td >Phosphate Buffered Saline free from Calcium and Magnesium (PBS)</td>
			<td >MEDIATECH</td>
			<td >21-040-CM</td>
			<td >Sterile</td>
		</tr>
		<tr >
			<td >S7975 Streptomycin Sulfate, 650-850U/mg</td>
			<td >USBIOLOGICAL</td>
			<td >3810-74-0</td>
			<td >Antibiotics</td>
		</tr>
		<tr >
			<td >Serological pipettes of 10mL or 25 mL</td>
			<td >SARSTEDT</td>
			<td >861254001</td>
			<td >Serological pipettes is used volumes higher than 1 mL</td>
		</tr>
		<tr >
			<td >Sodium Bicarbonate</td>
			<td >SIGMA-ALDRICH</td>
			<td >144-55-8</td>
			<td >pH correction for DMEM-F12</td>
		</tr>
		<tr >
			<td >Software Nis Elements Viewer</td>
			<td >NIKON</td>
			<td >Not applicable</td>
			<td >NIS-Elements Viewer is a free standalone program to view image files and datasets</td>
		</tr>
		<tr >
			<td >Sterile PBS and 2% of P/S solution</td>
			<td >LABORCRIN</td>
			<td >590338</td>
			<td >Add 1 mL of P/S in 40 mL of sterile PBS</td>
		</tr>
		<tr >
			<td >Straight iris scissors</td>
			<td >KATENA</td>
			<td >Not applicable</td>
			<td >Maintain in sterile beaker containing 70% ethanol solution</td>
		</tr>
		<tr >
			<td >Supernatant of L-929 cells</td>
			<td >Not applicable</td>
			<td >Not applicable</td>
			<td >L-929 is a lineage of mouse fibroblast cells used as a source of macrophage colony stimulating factor (M-CSF). L-929 supernatant was obtain from the protocol</td>
		</tr>
		<tr >
			<td >Surgical Scalpel Blade No.24 Stainless Steel</td>
			<td >SWANN-MORTON</td>
			<td >311</td>
			<td >Used for exposing epiphysis from bones</td>
		</tr>
		<tr >
			<td >Trypan Blue Solution, 0.4%</td>
			<td >GIBCO</td>
			<td >15250061</td>
			<td >Trypan Blue Solution, 0.4%, is routinely used as a cell stain to assess cell viability using the dye exclusion test</td>
		</tr>
		<tr >
			<td >Trypsin/EDTA solution 0,05%</td>
			<td >GIBCO</td>
			<td >25300-062</td>
			<td >Used to dissociate cells from culture bottle</td>
		</tr>
		<tr >
			<td >Water for Injection (WFI) for Cell Culture</td>
			<td >GIBCO</td>
			<td >A12873</td>
			<td >Sterile and endotoxin-free water</td>
		</tr>
	</tbody>
</table>

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.

Macrophages are large and adherent cells with special physiological characteristics. They show a diversity of morphologic presentations in culture because of the ability to adhere to glass and plastic, and their typical spread-out morphology is related to the emission of cytoplasmic extensions (Figure 1). Once bone marrow progenitors are exposed to M-CSF from L-929 cell supernatant and start the transformation to mature macrophages, they become adherent to the Petri plate.
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Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

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 ...

isolation-and-culture-of-bone-marrow-derived-macrophages-from-mice

Research

JoVE Journal

Immunology and Infection

2.9K Views.  Federal University of Minas Gerais. The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Video: Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

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

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

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

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

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

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

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

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

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 ...

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.

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 ...

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 various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

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 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 ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

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 ...