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/pubmed?term=((Tissue-resident+macrophages+originate+from+yolk-sac-derived+erythro-myeloid+progenitors%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((The+origin+and+kinetics+of+mononuclear+phagocytes%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">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/pubmed?term=((Macrophage+biology+in+development%2C+homeostasis+and+disease%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Origin+and+physiological+roles+of+inflammation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Macrophage+plasticity%2C+polarization%2C+and+function+in+health+and+disease%5BTitle%5D)+AND+%22Journal+of+Cellular+Physiology%22%5BJournal%5D)">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/pubmed?term=((Isolation+and+culture+of+murine+macrophages%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Culture+of+macrophage+colony-stimulating+factor+differentiated+human+monocyte-derived+macrophages%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Culture of macrophage colony-stimulating factor differentiated human monocyte-derived macrophages.</a> Journal of Visualized Experiments. (112), e54244(2016).</li>
<li>Gonçalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolation+and+characterization+of+murine+macrophages%5BTitle%5D)+AND+%22Current+Protocols+in+Immunology%22%5BJournal%5D)">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/pubmed?term=((Macrophages%3A+development+and+tissue+specialization%5BTitle%5D)+AND+%22Annual+Review+of+Immunology%22%5BJournal%5D)">Macrophages: development and tissue specialization.</a> Annual Review of Immunology. 33, 643-675 (2015).</li>
<li>Andrés, V., Pello, O. M., Silvestre-Roig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophage+proliferation+and+apoptosis+in+atherosclerosis%5BTitle%5D)+AND+%22Current+Opinion+in+Lipidology%22%5BJournal%5D)">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/pubmed?term=((Isolation%2C+culture%2C+and+polarization+of+murine+bone+marrow-derived+and+peritoneal+macrophages%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Biochemical+and+functional+characterization+of+three+activated+macrophage+populations%5BTitle%5D)+AND+%22Journal+of+Leukocyte+Biology%22%5BJournal%5D)">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., Andrés, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+macrophage+phagocytosis+assay%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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/pubmed?term=((Induction+of+tumoricidal+macrophages+from+bone+marrow+cells+of+normal+mice+or+mice+bearing+a+colony-stimulating-factor-producing+tumor%5BTitle%5D)+AND+%22Cancer+Immunology%2C+Immunotherapy%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">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/pubmed?term=((Differentiation+of+rat+bone+marrow+cells+into+macrophages+under+the+influence+of+mouse+L929+cell+supernatant%5BTitle%5D)+AND+%22Journal+of+Leukocyte+Biology%22%5BJournal%5D)">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/pubmed?term=((Bone+marrow-derived+macrophages+%28BMM%29%3A+isolation+and+applications%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+the+factor+in+L-cell+conditioned+medium+capable+of+stimulating+colony+formation+by+mouse+marrow+cells+in+culture%5BTitle%5D)+AND+%22Journal+of+Cellular+Physiology%22%5BJournal%5D)">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/pubmed?term=((Macrophage+colony-stimulating+factor+%28CSF-1%29%5BTitle%5D)+AND+%22Encyclopedia+of+Immunology%22%5BJournal%5D)">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/pubmed?term=((Two+physically%2C+functionally%2C+and+developmentally+distinct+peritoneal+macrophage+subsets%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+mouse+peritoneal+cavity+cells%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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><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&amp;eacute;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?</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&amp;prime;s Modified Eagle&amp;prime;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&lt;br/&gt; distilled water and add 3,7 g of sodium bicarbonate. Adjust pH to 7,2 and filter with 0,22 &amp;micro;M. Storage at 2 - 8 &amp;deg;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&amp;nbsp;</td><td>NIKON&amp;nbsp;</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 &amp;times; 20&amp;ndash;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 &amp;micro;M filter and store aliquots of 1.0 mL in 1.5 ml microfuge tubes in the freezer (-20&amp;deg;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

3.8K Views.  Federal University of Minas Gerais. Dit protocol is gericht op het verkrijgen van grote hoeveelheden niet-gestimuleerde macrofagen die zijn afgeleid van beenmerg en nooit zijn blootgesteld aan weefselfactoren of cytokines. Het belangrijkste voordeel van deze techniek is het aantal cellen dat van een enkel dier wordt verkregen. Met de juiste procedure kunnen ongeveer 2 keer 10 tot de macht 7 macrofagen worden verkregen.Deze methode zou onderzoekers kunnen helpen uit verschillende gebieden die macrofagen bestuderen, zoals ...

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

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

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 ...

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

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 ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

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 ...

Improved Protein Quantification Using Two-Step Mitochondrial Isolation

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Author Spotlight: Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial ...

Biology

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Source: Kozicky, L. et al., <a href="https://app.jove.com/v/56689">Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the activation of bone marrow-derived macrophages in response to bacterial lipopolysaccharide and intravenous immunoglobulin.

Source: Kozicky, L. et al., Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation. J. Vis. Exp. (2017)
In ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

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