This work was funded by the Canadian Institutes of Health Research (CIHR) (Grant MOP-119503) and the Natural Sciences and Engineering Council of Canada (NSERC). NSERC also supported summer studentships to Y.D. and A.A. YD is supported by the University of British Columbia (UBC) with a 4-year fellowship award, A.A is supported by CIHR with a graduate student Master&#39;s award (CGS-M). We thank Calvin Roskelley for assistance with the microscope used to generate the images in Figure 2. We also acknowledge support from the UBC Animal and Flow Cytometry Facilities.

Mice were euthanized in accordance with the Canadian Council on Animal Care guidelines for ethical animal research by procedures approved by the University of British Columbia Animal Care Committee.
1. Acquiring a Single Cell Bone Marrow Suspension from Mouse Femur and Tibia
<ol>
	<li>Switch on a biological safety cabinet (BSC) 15 min prior to start of procedure to purge cabinet air and allow for air flow stabilization, then clean/spray BSC surface with 70% ethanol. Keep everything as sterile as possible for the...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+(CSF)+and+macrophage+CSF-dependent+macrophage+phenotypes+display+differences+in+cytokine+profiles+and+transcription+factor+activities:+implications+for+CSF+blockade+in+inflammation.">Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation.</a> J Immunol. 178 (8), 5245-5252 (2007).</li><li>Lari, R., 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+lineage+phenotypes+and+osteoclastogenesis--complexity+in+the+control+by+GM-CSF+and.">Macrophage lineage phenotypes and osteoclastogenesis--complexity in the control by GM-CSF and.</a> Bone. 40 (2), 323-336 (2007).</li><li>Brasel, K., De Smedt, T., Smith, J. L., Maliszewski, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+murine+dendritic+cells+from+flt3-ligand-supplemented+bone+marrow+cultures.">Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures.</a> Blood. 96 (9), 3029-3039 (2000).</li><li>Angelov, G. S., Tomkowiak, M., Marcais, A., Leverrier, Y., Marvel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flt3+ligand-generated+murine+plasmacytoid+and+conventional+dendritic+cells+differ+in+their+capacity+to+prime+naive+CD8+T+cells+and+to+generate+memory+cells+in+vivo.">Flt3 ligand-generated murine plasmacytoid and conventional dendritic cells differ in their capacity to prime naive CD8 T cells and to generate memory cells in vivo.</a> J Immunol. 175 (1), 189-195 (2005).</li><li>Inaba, K., 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=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 176 (6), 1693-1702 (1992).</li><li>Lutz, M. B., 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=An+advanced+culture+method+for+generating+large+quantities+of+highly+pure+dendritic+cells+from+mouse+bone+marrow.">An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.</a> J Immunol Methods. 223 (1), 77-92 (1999).</li><li>Labeur, M. 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=Generation+of+tumor+immunity+by+bone+marrow-derived+dendritic+cells+correlates+with+dendritic+cell+maturation+stage.">Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.</a> J Immunol. 162 (1), 168-175 (1999).</li><li>Berthier, R., Martinon-Ego, C., Laharie, A. M., Marche, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-step+culture+method+starting+with+early+growth+factors+permits+enhanced+production+of+functional+dendritic+cells+from+murine+splenocytes.">A two-step culture method starting with early growth factors permits enhanced production of functional dendritic cells from murine splenocytes.</a> J Immunol Methods. 239 (1-2), 95-107 (2000).</li><li>Brasel, K., 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=Flt3+ligand+synergizes+with+granulocyte-macrophage+colony-stimulating+factor+or+granulocyte+colony-stimulating+factor+to+mobilize+hematopoietic+progenitor+cells+into+the+peripheral+blood+of+mice.">Flt3 ligand synergizes with granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor to mobilize hematopoietic progenitor cells into the peripheral blood of mice.</a> Blood. 90 (9), 3781-3788 (1997).</li><li>Segura, E., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+dendritic+cells+in+mice+and+humans.">Inflammatory dendritic cells in mice and humans.</a> Trends Immunol. 34 (9), 440-445 (2013).</li><li>West, M. 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=Enhanced+dendritic+cell+antigen+capture+via+toll-like+receptor-induced+actin+remodeling.">Enhanced dendritic cell antigen capture via toll-like receptor-induced actin remodeling.</a> Science. 305 (5687), 1153-1157 (2004).</li><li>Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M., Stockinger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+in+the+context+of+an+inflammatory+cytokine+milieu+supports+de+novo+differentiation+of+IL-17-producing+T+cells.">TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.</a> Immunity. 24 (2), 179-189 (2006).</li><li>Goodridge, H. 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=Activation+of+the+innate+immune+receptor+Dectin-1+upon+formation+of+a+'phagocytic+synapse.">Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse.</a> Nature. 472 (7344), 471-475 (2011).</li><li>Shalek, A. K., 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=Single-cell+RNA-seq+reveals+dynamic+paracrine+control+of+cellular+variation.">Single-cell RNA-seq reveals dynamic paracrine control of cellular variation.</a> Nature. 510 (7505), 363-369 (2014).</li><li>Vander Lugt, B., 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=Transcriptional+programming+of+dendritic+cells+for+enhanced+MHC+class+II+antigen+presentation.">Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation.</a> Nat Immunol. 15 (2), 161-167 (2014).</li><li>Shibata, Y., 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=GM-CSF+regulates+alveolar+macrophage+differentiation+and+innate+immunity+in+the+lung+through+PU.1.">GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1.</a> Immunity. 15 (4), 557-567 (2001).</li><li>Lacey, D. C., 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=Defining+GM-CSF-+and+Macrophage-CSF+Dependent+Macrophage+Responses+by+In+Vitro+Models.">Defining GM-CSF- and Macrophage-CSF Dependent Macrophage Responses by In Vitro Models.</a> J Immunol. 188 (11), 5752-5765 (2012).</li><li>Poon, G. F., 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=Hyaluronan+Binding+Identifies+a+Functionally+Distinct+Alveolar+Macrophage-like+Population+in+Bone+Marrow-Derived+Dendritic+Cell+Cultures.">Hyaluronan Binding Identifies a Functionally Distinct Alveolar Macrophage-like Population in Bone Marrow-Derived Dendritic Cell Cultures.</a> J Immunol. 195 (2), 632-642 (2015).</li><li>Helft, J., 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=GM-CSF+Mouse+Bone+Marrow+Cultures+Comprise+a+Heterogeneous+Population+of+CD11c(+)MHCII(+)+Macrophages+and+Dendritic+Cells.">GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells.</a> Immunity. 42 (6), 1197-1211 (2015).</li><li>Stockinger, B., Zal, T., Zal, A., Gray, D. 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The authors have no financial conflicts of interest.

In this manuscript, we provide a method for generating GM-CSF derived macrophages and DCs from a single mouse bone marrow culture that is adapted from Lutz et al.6. MHCII&#160; expression and FL-HA binding distinguishes between immature DCs and macrophages in this culture (see Figure 3C), which previously has been difficult. This, together with another report by Helft et al.19, demonstrates heterogeneity within GM-CSF induced BMDC cultures that were previously thou...

Several in vitro culture methods have been described to generate bone marrow-derived macrophages (BMDMs) and bone marrow-derived DCs (BMDCs) using one or a combination of growth factors. BMDMs can be generated by culturing bone marrow cells using either macrophage colony stimulating factor (M-CSF) or GM-CSF1,2. For BMDCs, the addition of FLT3 ligand to the bone marrow culture gives rise to non-adherent classical and plasmacytoid DCs (CD11chigh/MHCIIhigh and CD11clo, B220+ respectively) after 9 days in culture3,4. In contrast, non-adherent cells generated after 7 to 10 days in culture with GM-CSF alone5,6, GM-CSF and IL-47, or GM-CSF and FLT3 ligand8,9 generate BMDCs more closely resembling inflammatory DCs (CD11chigh, MHCIIhigh CD11b+)10. While these in vitro cultures are used to generate macrophages or DCs, it is unclear if each culture gives rise to pure populations. For example, although adherent cells in the GM-CSF cultures are described to be macrophages5, the non-adherent cells from the same culture are used as DCs6,11-13, with the presumption that they are homogeneous and any observed variability is due to different stages of development14,15. Furthermore, studies have found GM-CSF to be an essential growth factor for alveolar macrophage development in vivo16,17, and can be used in vitro to generate alveolar-like macrophages16,17,18.
Other than adherence, the procedures for generating macrophages and DCs from GM-CSF treated bone marrow cultures are very similar suggesting heterogeneity may exist within GM-CSF bone marrow cultures. This indeed seems to be the case as two papers report the presence of BMDMs in the non-adherent fraction of BMDC cultures. In one paper, they identified a population of cells as CD11c+, CD11b+, MHCIImid, MerTK+, and CD115+, which expressed a gene expression signature that most closely resembled alveolar macrophages and had a reduced ability to activate T cells19. The second paper used MHCII and FL-HA to identify an alveolar macrophage-like population (CD11c+, MHCIImid/low, FL-HAhigh) that was distinct from immature (CD11c+, MHCIImid, FL-HAlow) and mature DCs (CD11c+, MHCIIhigh), both phenotypically and functionally18. These papers both illustrate that GM-CSF BMDC cultures are heterogeneous, containing both macrophage and DC populations indicating that care should be taken when interpreting data from BMDC cultures.
This protocol describes how to isolate bone marrow, culture bone marrow cells in GM-CSF, and identify the alveolar macrophage-like population from the immature and mature DCs in the bone marrow culture by flow cytometry using FL-HA binding and MHCII expression. This procedure is based on the established procedure of Lutz et al.6 and is able to generate 5 - 10 x 106 non-adherent cells on day 7 of a 10 ml culture. The culture is usable from days 7 to 10 and yields a heterogeneous population of macrophages, immature and mature DCs, as well as some progenitors at day 7. This provides a simple method to grow and isolate in vitro alveolar-like macrophages in large quantities.

<table>
	<tbody>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD&#160;</td>
			<td>LSR-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Inverted Microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>DMI4000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Thermo Fisher</td>
			<td>ST-40R</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>Nuaire</td>
			<td>NU-425-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 ml</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>26 1/2 Gauge Needle</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Conical Tube&#160;</td>
			<td>Corning</td>
			<td>357070</td>
			<td>*Falcon brand</td>
		</tr>
		<tr>
			<td>Eppendorf tubes (1.5 ml)</td>
			<td>Corning</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polystyrene round bottomed tubes</td>
			<td>Corning</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Tools</td>
			<td>Fine Science Tools&#160;</td>
			<td>*Various&#160;</td>
			<td>*Dissection scissors, dumont forcep and standard forcep&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer&#160;</td>
			<td>Richert</td>
			<td>1490</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 100 x 15 mm Petri Dish</td>
			<td>Corning</td>
			<td>351029</td>
			<td>*Falcon brand</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo Fisher</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>BDH</td>
			<td>BDH0208-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Bioreagents</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>B7651-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134-1KG</td>
			<td>Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Thermo Fisher</td>
			<td>14175-095&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher</td>
			<td>15630-080</td>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G8540-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS Ultrapure</td>
			<td>Invivogen</td>
			<td>tlrl-3pelps</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100x</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>BDH</td>
			<td>BDH0268-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>BDH</td>
			<td>BDH9258-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant GM-CSF</td>
			<td>Peprotech</td>
			<td>315-03-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Rooster Comb Sodium Hyaluronate&#160;</td>
			<td>Sigma</td>
			<td>H5388-1G</td>
			<td>*Used to make fluoresceinated hyaluronan</td>
		</tr>
		<tr>
			<td>RPMI-1640&#160;</td>
			<td>Thermo Fisher</td>
			<td>21870-076</td>
			<td>No sodium pyruvate no glutamine. Warm media to 37oC before using.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher&#160;</td>
			<td>5271-10&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma</td>
			<td>50876-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma</td>
			<td>P5290-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Fisher Bioreagents</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Fc Receptor (unlabeled), Tissue Culture Supernatant</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Clone: 2.4G2</td>
		</tr>
		<tr>
			<td>Anti-CD11c PeCy7</td>
			<td>eBioscience</td>
			<td>25-0114-82</td>
			<td>Clone: N418</td>
		</tr>
		<tr>
			<td>Anti-Gr-1 efluor450</td>
			<td>eBioscience</td>
			<td>48-5931-82</td>
			<td>Clone: RB6-8C5</td>
		</tr>
		<tr>
			<td>Anti-MHCII APC</td>
			<td>eBioscience</td>
			<td>17-5321-82</td>
			<td>Clone: M5/114.15.2</td>
		</tr>
		<tr>
			<td>Biotinylated Anti-MerTK</td>
			<td>Abcam</td>
			<td>BAF591</td>
			<td>Goat polyclonal IgG</td>
		</tr>
		<tr>
			<td>Streptavidin PE</td>
			<td>eBioscience</td>
			<td>12-4317-87</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma</td>
			<td>P4170-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td>Sigma</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and identification of gm-csf derived alveolar-like macrophages and dendritic cells from mouse bone marrow

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

A flowchart summarizing the major steps of this method is shown in Figure 1. The density and morphology of the bone marrow culture at different times of culture are shown in Figure 2. At day 1, the cells are small and sparse but by day 3, there are more cells, some are larger and a few have begun to adhere. By day 6 there is a definite adherent and non-adherent fraction (Figure 2A). The culture can be harvested from day 7 - 10 with a high...

Watch this Scientific Journal Video about Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow at JoVE.com

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

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

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

The overall goal is this procedure is to generate and distinguish alveolar-like macrophages from dendritic cells in in vitro murine bone marrow cell cultures supplemented with GM-CSF. This in vitro method can help answer key questions regarding the function of dendritic cells or macrophages. The main advantage of the technique is that it can generate sufficient cell numbers and it can distinguish between dendritic cells and alveolar-like macrophages.The implications of this technique may extend towards the therapy of pulmonary proteinosis as it facilitates the acquisition of alveolar-like macrophages, which can be used to treat this disease. Generally, individuals new to this method will need practice with isolating the cleaned bones and flushing the bone marrow. Visual demonstration of this method is useful as not all techniques used as easily understood by written instruction alone.15 minutes before beginning the dissection, turn on a biological safety cabinet to purge the cabinet air, and to stabilize the air flow, and clean the cabinet surface with 70%ethanol. When the cabinet is ready, place a mouse on top of one to two paper towels in the prone position and spray the animal with 70%ethanol. Then, with the non-dominant hand, lift the skin around the thoracic area and use scissors to make an incision.Grasping both ends of the incision, carefully pull until the two ends of the incision meet at the stomach. Then place the mouse in the supine position and use one hand to pull the lower half of the skin down until the legs are exposed. Holding the foot, cut the Achilles tendons above the ankle joint and the ligaments connecting the muscles to the foot at the lower end of the tibia to loosen the foot from the leg bones.Keeping the leg raised, remove the muscles and ligaments around the knee joint at the lower end of the femur to severe the thigh muscles from the lower leg. Using forceps, gently pull the loosened muscles away from the fibular and femoral surfaces. Then pressing the scissors in the open position at the hip joint, rotate the leg and gently pull on the femur to separate the leg bone from the hip joint while cutting the leg free from the body.Now holding the leg at the hip end with a sterile forceps, use another forceps to remove the foot from the ankle end. Then holding the distal end of the femur with one forceps and the proximal end of the tibia and fibula with another, carefully bend the tibia and fibula in the opposite direction of the knee joint to separate the lower leg from the femur and patella. Use the forceps to remove the remaining ligaments, muscles, and fibula from the lower leg bones and place the cleaned tibia on an inverted Petri dish lid, containing one to two milliliters of HBSS/FCS.Holding the lower end of the femur with one set of forceps and the patella with another, bend the knee and surrounding tissues in the opposite direction to remove the joint. Then remove any of the remaining connective tissues from the femur and place the bone on the inverted Petri dish lid. When all of the bones have been harvested, use forceps to clean any residual tissue still attached to the bones and immerse the bones in 10 milliliters of HBSS/FCS within the base of the Petri dish.Keeping the dish partially shielded with a sterile lid, cut each of the cleaned bones in half with scissors. Then use a one-milliliter syringe, equipped with a 26.5-gauge needle to flush one milliliter of HBSS/FCS from the dish into the end of each bone piece until all of the red marrow has been liberated. Pass any visible clumps of bone marrow through the syringe several times to form a single cell suspension, followed by thorough mixing with a 10 milliliter pipette.Transfer the cells to a collection tube and rinse the Petri dish one time with five milliliters of HBSS/FCS to collect any residual cells. Then pool the wash in the collection tube and place the cells on ice. To set up a bone marrow cell culture, centrifuge the collected cells and aspirate the supernatant with a sterile glass Pasteur pipette attached to vacuum suction.Loosen the red blood cell-rich pellet with tapping. Then add 10 milliliters of RBC lysis buffer and resuspend the cells with vortexing. After a five-minute, room temperature incubation, arrest the lysis with 10 milliliters of HBSS/FCS and spin down the cells, resuspending the pellet in bone marrow cell medium.Count the number of viable cells by Trypan Blue exclusion and transfer the needed cells into a new tube with bone marrow cell medium. Then collect the cells by centrifugation and resuspend the pellet at a four million cell per milliliter dilution in bone marrow cell medium with 20 nanograms per milliliter of GM-CSF. Next, add 9.5 milliliters of bone marrow cell medium supplemented with 20 nanograms per milliliter of GM-CSF to one sterile bacterial Petri dish per culture and add 500 microliters of cells to the center of each dish.Incubate the cells at 37 degrees Celsius and five percent CO2. On day three, add 10 milliliters of fresh bone marrow medium supplemented with 20 nanograms per milliliter of GM-CSF to each culture, taking care to minimize any disturbances to the cells. On day six, carefully transfer 10 milliliters of cell culture supernatant to a sterile 50 milliliter conical tube.Centrifuge the cells and resuspend the pellet in 10 milliliters of fresh bone marrow cell medium, containing 20 nanograms per milliliter of GM-CSF. Then gently vortex the cell suspension and return the cells to the culture dish. On day one, the bone marrow cells are small and sparse but by day three, the cells have increased in number and size, and some have begun to adhere.By day six, there are more cells and both adherent and non-adherent fractions are observed. The culture can be harvested from day seven to 10 and gated by size and live cells with a higher percentage of CD11c-positive cells obtained from day 10 cultures. At day seven, the collected non-adherent fraction is greatly enriched for cells with a dendritic morphology.The CD11c-positive, GR1-negative populations, harvested on day seven and 10, can be divided into three main populations. Macrophages, immature dendritic cells, and mature dendritic cells by MHCII expression and FL-HA binding. Interestingly, MerTK, although considered to be a macrophage marker, exhibits a robust expression on both macrophage and immature dendritic cell populations, harvested on days seven and 10, with a low level of expression observed on mature dendritic cells.While attempting this procedure, it is important to remember to use sterile techniques throughout the bone marrow harvest and cell culture. Following this procedure, the cells also can be sorted to examine cell populations separately, or they can be stimulated with inflammatory agents and subsequently analyzed for their cytokine production by intracellular staining and flow cytometry. After watching this video, you should have a good understanding of how to isolate bone marrow cells and culture them in GM-CSF to generate both dendritic cells and alveolar-like macrophages.

generation-identification-gm-csf-derived-alveolar-like-macrophages

Research

JoVE Journal

Immunology and Infection

17.8K 조회수.  University of British Columbia. The overall goal is this procedure is to generate and distinguish alveolar-like macrophages from dendritic cells in in vitro murine bone marrow cell cultures supplemented with GM-CSF. This in vitro method can help answer key questions regarding the function of dendritic cells or macrophages. The main advantage of the technique is that it can generate sufficient cell numbers and it can distinguish between dendritic cells and alveolar-like macrophages.The implications of this technique m...