Name: expression of fluorescent fusion proteins in murine bone marrow-derived dendritic cells and macrophages
Uploaded: 2018-10-30T12:30:00
Description: Watch this Scientific Journal Video about Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages at JoVE.com

This work was supported by Czech Science Foundation (GACR) (project number 16-07425S), by Charles University Grant Agency (GAUK) (project number 923116) and by institutional funding from the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic (RVO 68378050).

All methods described here have been approved by the Expert Committee on the Welfare of Experimental Animals of the Institute of Molecular Genetics and by the Academy of Sciences of the Czech Republic.
1. Reagent Preparation
<ol>
	<li>Prepare the ammonium-chloride-potassium (ACK) buffer. Add 4.145 g of NH4Cl and 0.5 g of KHCO3 to 500 mL of ddH2O, then add 100 &#181;L of 0.5 M ethylenediaminetetraacetic acid (EDTA) and filter-sterilize.</li>
	<li>Prepare polye...

<ol>
	<li>Moghaddam, A. 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=Macrophage+plasticity,+polarization+and+function+in+health+and+disease.">Macrophage plasticity, polarization and function in health and disease.</a> Journal of Cellular Physiology. , (2018).</li><li>Qian, C., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+in+the+regulation+of+immunity+and+inflammation.">Dendritic cells in the regulation of immunity and inflammation.</a> Seminars in Immunology. 35, 3-11 (2018).</li><li>Amulic, B., Cazalet, C., Hayes, G. L., Metzler, K. D., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+function:+from+mechanisms+to+disease.">Neutrophil function: from mechanisms to disease.</a> Annual Review of Immunology. 30, 459-489 (2012).</li><li>Kondo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid+and+myeloid+lineage+commitment+in+multipotent+hematopoietic+progenitors.">Lymphoid and myeloid lineage commitment in multipotent hematopoietic progenitors.</a> Immunological Reviews. 238 (1), 37-46 (2010).</li><li>Andrews, T., Sullivan, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+in+patients+with+inherited+defects+in+phagocytic+function.">Infections in patients with inherited defects in phagocytic function.</a> Clinical Microbiology Reviews. 16 (4), 597-621 (2003).</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>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>Scheicher, 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=Recombinant+GM-CSF+induces+in+vitro+differentiation+of+dendritic+cells+from+mouse+bone+marrow.">Recombinant GM-CSF induces in vitro differentiation of dendritic cells from mouse bone marrow.</a> Advances in Experimental Medicine and Biology. 329, 269-273 (1993).</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=The+macrophage+colony-stimulating+factor,+CSF-1.">The macrophage colony-stimulating factor, CSF-1.</a> Methods in Enzymology. , 564-587 (1985).</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, (2008).</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> Journal of Immunological Methods. 223 (1), 77-92 (1999).</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> Journal of Experimental Medicine. 176 (6), 1693-1702 (1992).</li><li>Chamberlain, L. M., Godek, M. L., Gonzalez-Juarrero, M., Grainger, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+non-equivalence+of+murine+(monocyte-)+macrophage+cells+in+biomaterial+and+inflammatory+models.">Phenotypic non-equivalence of murine (monocyte-) macrophage cells in biomaterial and inflammatory models.</a> Journal of Biomedical Materials Research Part A. 88 (4), 858-871 (2009).</li><li>Remington, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein:+a+perspective.">Green fluorescent protein: a perspective.</a> Protein Science. 20 (9), 1509-1519 (2011).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+In+Vivo+Imaging+Using+Fluorescent+Proteins.">Strategies for In Vivo Imaging Using Fluorescent Proteins.</a> Journal of Cellular Biochemistry. 118 (9), 2571-2580 (2017).</li><li>Telford, W. G., Hawley, T., Subach, F., Verkhusha, V., Hawley, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+fluorescent+proteins.">Flow cytometry of fluorescent proteins.</a> Methods. 57 (3), 318-330 (2012).</li><li>Zhang, X., Edwards, J. P., 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+expression+of+exogenous+genes+in+macrophages:+obstacles+and+opportunities.">The expression of exogenous genes in macrophages: obstacles and opportunities.</a> Methods in Molecular Biology. , 123-143 (2009).</li><li>Zal, T., Volkmann, A., 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=Mechanisms+of+tolerance+induction+in+major+histocompatibility+complex+class+II-restricted+T+cells+specific+for+a+blood-borne+self-antigen.">Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen.</a> Journal of Experimental Medicine. 180 (6), 2089-2099 (1994).</li><li>Takeshita, S., Kaji, K., Kudo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Characterization+of+the+New+Osteoclast+Progenitor+with+Macrophage+Phenotypes+Being+Able+to+Differentiate+into+Mature+Osteoclasts.">Identification and Characterization of the New Osteoclast Progenitor with Macrophage Phenotypes Being Able to Differentiate into Mature Osteoclasts.</a> Journal of Bone and Mineral Research. 15 (8), 1477-1488 (2000).</li><li>Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. 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+pCL+vector+system:+rapid+production+of+helper-free,+high-titer,+recombinant+retroviruses.">The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.</a> Journal of Virology. 70 (8), 5701-5705 (1996).</li><li>Janssen, E., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptor+proteins+in+lymphocyte+activation.">Adaptor proteins in lymphocyte activation.</a> Current Opinion in Immunology. 15 (3), 269-276 (2003).</li><li>Ferguson, P. J., Laxer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+discoveries+in+CRMO:+IL-1beta,+the+neutrophil,+and+the+microbiome+implicated+in+disease+pathogenesis+in+Pstpip2-deficient+mice.">New discoveries in CRMO: IL-1beta, the neutrophil, and the microbiome implicated in disease pathogenesis in Pstpip2-deficient mice.</a> Seminars in Immunopathology. 37 (4), 407-412 (2015).</li><li>Holleman, 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=Expression+of+the+outcome+predictor+in+acute+leukemia+1+(OPAL1)+gene+is+not+an+independent+prognostic+factor+in+patients+treated+according+to+COALL+or+St+Jude+protocols.">Expression of the outcome predictor in acute leukemia 1 (OPAL1) gene is not an independent prognostic factor in patients treated according to COALL or St Jude protocols.</a> Blood. 108 (6), 1984-1990 (1984).</li><li>Zjablovskaja, P., Danek, P., Kardosova, M., Alberich-Jorda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+Differentiation+of+Murine+Myeloid+Precursor+32D/G-CSF-R+Cells.">Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells.</a> Journal of Visualized Experiments: JoVE. (132), (2018).</li><li>Kralova, 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=The+Transmembrane+Adaptor+Protein+SCIMP+Facilitates+Sustained+Dectin-1+Signaling+in+Dendritic+Cells.">The Transmembrane Adaptor Protein SCIMP Facilitates Sustained Dectin-1 Signaling in Dendritic Cells.</a> Journal of Biological Chemistry. 291 (32), 16530-16540 (2016).</li><li>Maess, M. B., Wittig, B., Lorkowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+of+human+THP-1+macrophages+by+nucleofection.">Highly efficient transfection of human THP-1 macrophages by nucleofection.</a> Journal of Visualized Experiments: JoVE. (91), e51960 (2014).</li><li>Bowles, R., Patil, S., Pincas, H., Sealfon, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocol+for+efficient+transfection+of+dendritic+cells+without+cell+maturation.">Optimized protocol for efficient transfection of dendritic cells without cell maturation.</a> Journal of Visualized Experiments: JoVE. (53), e2766 (2011).</li><li>Siegert, I., 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=Electroporation+of+siRNA+into+mouse+bone+marrow-derived+macrophages+and+dendritic+cells.">Electroporation of siRNA into mouse bone marrow-derived macrophages and dendritic cells.</a> Methods in Molecular Biology. 1121, 111-119 (2014).</li><li>Lee, C. 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=Adenovirus-Mediated+Gene+Delivery:+Potential+Applications+for+Gene+and+Cell-Based+Therapies+in+the+New+Era+of+Personalized+Medicine.">Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine.</a> Genes &amp; Diseases. 4 (2), 43-63 (2017).</li><li>Jin, L., Zeng, X., Liu, M., Deng, Y., He, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+progress+in+gene+delivery+technology+based+on+chemical+methods+and+nano-carriers.">Current progress in gene delivery technology based on chemical methods and nano-carriers.</a> Theranostics. 4 (3), 240-255 (2014).</li><li>Muruve, D. 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=The+inflammasome+recognizes+cytosolic+microbial+and+host+DNA+and+triggers+an+innate+immune+response.">The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response.</a> Nature. 452 (7183), 103-107 (2008).</li><li>Yang, Y., Li, Q., Ertl, H. C., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+humoral+immune+responses+to+viral+antigens+create+barriers+to+lung-directed+gene+therapy+with+recombinant+adenoviruses.">Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.</a> Journal of Virology. 69 (4), 2004-2015 (1995).</li><li>Tallone, T., 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=A+mouse+model+for+adenovirus+gene+delivery.">A mouse model for adenovirus gene delivery.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (14), 7910-7915 (2001).</li><li>Milone, M. C., O'Doherty, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+use+of+lentiviral+vectors.">Clinical use of lentiviral vectors.</a> Leukemia. , (2018).</li><li>McTaggart, S., Al-Rubeai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+vectors+for+human+gene+delivery.">Retroviral vectors for human gene delivery.</a> Biotechnology Advances. 20 (1), 1-31 (2002).</li><li>Gibson, T. J., Seiler, M., Veitia, R. 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+transience+of+transient+overexpression.">The transience of transient overexpression.</a> Nature Methods. 10 (8), 715-721 (2013).</li></ol>

The expression of protein of interest in target cells is a key step in many types of biological studies. Differentiated macrophages and dendritic cells are difficult to transfect by standard transfection and retroviral transduction techniques. Bypassing the transfection of these differentiated cells with retroviral transduction of bone marrow progenitors, followed by differentiation when they already carry the desired construct, is a critical step allowing the expression of ectopic cDNAs in these cell types. An example o...

Myeloid cells represent an indispensable part of our defense mechanisms against pathogens. They are able to rapidly eliminate microbes, as well as dying cells. In addition, they are also involved in regulating tissue development and repair and in maintaining homeostasis1,2,3. All myeloid cells differentiate from common myeloid progenitors in the bone marrow. Their differentiation into many functionally and morphologically distinct subsets is to a large extent controlled by cytokines and their various combinations4. The most intensively studied myeloid cell subsets include neutrophilic granulocytes, macrophages and dendritic cells. Defects in any of these populations lead to potentially life-threatening consequences and cause severe dysfunctions of the immune system in humans and mice1,2,3,5,6.
Unlike neutrophilic granulocytes, dendritic cells and macrophages are tissue resident cells and their abundance in immune organs is relatively low. As a result, the isolation and purification of primary dendritic cells and macrophages for experiments requiring a large number of these cells is expensive and often impossible. To solve this problem, protocols have been developed to obtain large amounts of homogenous macrophages or dendritic cells in vitro. These approaches are based on the differentiation of murine bone marrow cells in the presence of cytokines: macrophage colony-stimulating factor (M-CSF) for macrophages and granulocyte-macrophage colony-stimulating factor (GM-CSF) or Flt3 ligand for dendritic cells7,8,9,10,11,12. Cells generated by this method are commonly described in the literature as bone marrow derived macrophages (BMDMs) and bone marrow derived dendritic cells (BMDCs). They have more physiological properties in common with primary macrophages or dendritic cells than with corresponding cell lines. Another major advantage is the possibility of obtaining these cells form genetically modified mice13. Comparative studies between wild-type cells and cells derived from genetically modified mice are often critical for uncovering novel functions of genes or proteins of interest.
Analysis of subcellular localization of proteins in living cells requires the coupling of a fluorescent label to the protein of interest in vivo. This is most commonly achieved by expressing genetically encoded fusion construct composed of an analyzed protein coupled (often via a short linker) to a fluorescent protein (e.g., green fluorescent protein (GFP))14,15,16. The expression of fluorescently tagged proteins in dendritic cells or macrophages is challenging. These cells are generally difficult to transfect by standard transfection procedures and the efficiencies tend to be very low. Moreover, the transfection is transient, it generates cellular stress and achieved intensity of fluorescence might not be sufficient for microscopy17. In order to obtain a reasonable fraction of these cells with a sufficient level of transgene expression, the infection of bone marrow progenitor cells with retroviral vectors and their subsequent differentiation into BMDMs or BMDCs has become a very efficient approach. It has allowed for the analysis of the proteins of myeloid origin in their native cellular environment, both in a steady state or during processes that are critical for immune response such as phagocytosis, immunological synapse formation or migration. Here, we describe a protocol that allows stable expression of fluorescently tagged proteins of interest in murine bone marrow derived macrophages and dendritic cells.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>15028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>10270</td>
			<td>For media suplementation</td>
		</tr>
		<tr>
			<td>KHCO3</td>
			<td>Lachema, Brno, Czech Republic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>BB Pharma AS, Prague, Czech Republic</td>
			<td>N/A</td>
			<td>PENICILIN G 1,0 DRASELN&#193; SOL&#39; BIOTIKA</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>S9137</td>
			<td>Streptomycin sulfate salt powder</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Dr. Kulich Pharma, Hradec Kr&#225;lov&#233;, Czech Republic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, linear, MW 25,000</td>
			<td>Polyscience, Warrington, PA, USA</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich (Merck, Kenilworth, NJ, USA)</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Prepared in-house by media facility of IMG ASCR, Prague, Czech Republic</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse/human CD11b Antibody, clone M1/70</td>
			<td>BioLegend (San Diego, CA, USA)</td>
			<td>101212</td>
			<td>flow cytometry analysis</td>
		</tr>
		<tr>
			<td>PE anti-mouse F4/80 Antibody, clone BM8</td>
			<td>BioLegend (San Diego, CA, USA)</td>
			<td>123110</td>
			<td>flow cytometry analysis</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD11c Antibody, clone N418</td>
			<td>BioLegend (San Diego, CA, USA)</td>
			<td>117310</td>
			<td>flow cytometry analysis</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>PeproTech (Rocky Hill, NJ, USA)</td>
			<td>315-02</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>PeproTech (Rocky Hill, NJ, USA)</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33258</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>H1398</td>
			<td>flow cytometry analysis use at 1-2 &#181;g/ml</td>
		</tr>
	</tbody>
</table>

expression of fluorescent fusion proteins in murine bone marrow-derived dendritic cells and macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

Signaling adaptor proteins are usually small proteins without any enzymatic activity. They possess various interaction domains or motifs, which mediate binding to other proteins involved in signal transduction, including tyrosine kinases, phosphatases, ubiquitin ligases and others21. For the demonstration of the functionality of this protocol myeloid cell adaptors PSTPIP2 and OPAL1 were selected. PSTPIP2 is a well characterized protein involved in the regulation of...

Watch this Scientific Journal Video about Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages at JoVE.com

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Research

JoVE Journal

Biology

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...