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...

<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>

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.
<p class="jove...

Watch this Scientific Journal Video about Isolation and Culture of Bone Marrow-Derived Macrophages from Mice at JoVE.com

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 ...

This protocol aims to obtain high amounts of unstimulated macrophages that have been derived from bone marrow and have never been exposed to tissue factors or cytokines. The main advantage of this technique is the number of cells acquired from a single animal. With the right procedure, approximately 2 times 10 to the power of 7 macrophages can be obtained.This method could help researchers from different areas studying macrophages such as cell biology, pathology, immunology, and parasitology, Because cells are always susceptible to contamination, being careful with all sterile steps and using a laminar flow biosafety cabinet are necessary to maintain cell viability. Demonstrating the procedure will be Izabela Aparecida de Souza. A Master degree student from our laboratory.To begin the procedure on the properly euthanized eight week old C57BL/6 wild type male mouse, soak the mouse with 70%ethanol solution. Using sterile scissors, make a one centimeter incision along the abdomen and remove the skin until the muscle of the hind legs is fully exposed. Then carefully remove the hind legs at the height of the hip without breaking the femur and tibia.Put the intact hind legs in a conical centrifuge tube containing 70%ethanol solution. Inside a laminar flow biosafety cabinet, transfer the isolated legs from 70%ethanol to sterile PBS. Using forceps and disinfectant wipes, clean the bones by removing all attached muscles and fascia.Then, use a sterile surgical scalpel blade to cut the bone epiphysis on both ends, exposing the bone marrow. Fill a 20-milliliter syringe with 10 milliliters of sterile PBS supplemented with 2%penicillin streptomycin solution, and connect a 26-gauge needle to it. Hold the bone using anatomical dissection forceps with one hand.Use the other hand to insert the needle into the bone cavity carefully without crushing the bone. Then, wash out the inside of the bone with PBS and collect the bone marrow in a sterile conical centrifuge tube. Once washed, the bone cavity should appear white.Centrifuge the collected cells at 300G and four degrees Celsius for 10 minutes. Discard the supernatant and resuspend the cell pellet in one milliliter of DMEM/F1210 medium. Homogenize the suspension and add another nine milliliters of the medium to bring the total volume up to 10 milliliters.Add one milliliter of the progenitor cell suspension into each of 10 round plastic Petri dishes, measuring 100 milliliters by 20 millimeters. Spread the cells across the plates with a pipette to obtain a uniform distribution. Then, add nine milliliters of DMEM/F1210 supplemented with 20%of L929 cell supernatant to each dish.Incubate the dishes at 37 degrees Celsius and 5%carbon dioxide. On the third day, add 10 milliliters of DMEM/F1210 medium supplemented with 20%of L929 cell supernatant to each dish. The resulting 20 milliliters of culture medium in each dish is sufficient for cell growth until maturation.Discard the supernatants from all culture dishes. Wash each culture dish with 10 milliliters of sterile magnesium and calcium free PBS, prewarmed to 37 degrees Celsius, and discard the solution after washing. Add three milliliters of non-enzymatic cell dissociation solution, prewarmed to 37 degrees Celsius to each dish.Incubate for 10 minutes at 37 degrees Celsius and 5%carbon dioxide. Then, use an inverted microscope to visualize whether the macrophages dissociate from the cell culture dishes. Using a serological pipette wash the dishes with the non-enzymatic cell dissociation solution repeatedly performing circular movements to ensure complete macrophage dissociation.Collect and combine the solution from all the culture dishes into a 50 milliliter conical centrifuge tube. Once more, add 10 milliliters of sterile, prewarmed PBS to the empty culture dishes. Centrifuge the conical tube containing the combined collected solution at 300G and four degrees Celsius for 10 minutes.Discard the supernatant and resuspend the pellet with one milliliter of DMEM/F1210. Homogenize the suspension slowly and carefully by pipetting it up and down without damaging the cells. Dilute a 10 microliter aliquot of the macrophage solution by adding 10 microliters of trypan blue to count the cells using a hemocytometer.At day seven, each dish would produce approximately 2 million macrophages. Exposure to macrophage colony stimulating factor from L929 supernatant gradually transformed the bone marrow progenitors to mature macrophages, displaying typical spread out morphology due to cytoplasmic extensions. On day three, a few immature macrophages have appeared, having a typical round shape morphology with few membrane projections.Although they transformed along the entire process, seven days were necessary to achieve an adequate number and maturity. The obtained macrophages displayed a homogenous population in terms of size and granularity in the flow cytometry analysis. Furthermore, 100%of the population expressed the characteristic F480 and CD11B surface molecules, forming a single well-defined population of cells.The phagocytosis capability of the mature and well differentiated macrophages was confirmed by fluorescence microscopy images showing phagocytose Leishmania major parasites inside the macrophages. The legs should be removed from the animal carefully because it is performed in a non-sterile environment and is the most important source of contamination. Legs must not be broken outside of the laminar flow biosafety cabinet.The bone marrow derived macrophages obtained from this protocol can be used to subsequently perform macrophage in vitro polarization to understand macrophage physiology.

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JoVE Journal

Immunology and Infection

2.1K Views.  Federal University of Minas Gerais. This protocol aims to obtain high amounts of unstimulated macrophages that have been derived from bone marrow and have never been exposed to tissue factors or cytokines. The main advantage of this technique is the number of cells acquired from a single animal. With the right procedure, approximately 2 times 10 to the power of 7 macrophages can be obtained.This method could help researchers from different areas studying macrophages such as cell biology, pathology, immunology, and parasi...