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 polyethylenimine (PEI) solution. Add 0.1 g of PEI to 90 mL of ddH2O. While stirring, add 1 M HCl dropwise until the pH is lower than 2.0. Stir for up to 3 h until PEI is dissolved and then adjust pH to 7.2 with 1 M NaOH. Adjust the volume to 100 mL with ddH2O and filter-sterilize. Make 1&#8211;2 mL aliquots and store at -20 &#176;C. 
	NOTE: After thawing, PEI can be stored at 4 &#176;C for up to 2 weeks but should not be re-frozen.</li>
	<li>Prepare cell culture supernatants containing M-CSF or GM-CSF. These supernatants can be made in advance and stored in -80 &#176;C. To make these supernatants, grow the cytokine-producing cells (J558 cells for GM-CSF 18 or CMG 14-12 cells for M-CSF 19) in a 10 cm Petri dish in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% fetal bovine serum (FBS) to confluence. Then transfer all cells to 200 mL of media in T150 tissue culture flask and culture for additional 4 days at 5% CO2/37 &#176;C. Collect the supernatant and filter over 0.2 &#181;m sterilization filter. Make aliquots and store these at -80 &#176;C.</li>
	<li>Prepare 100 mL of cell culture medium: DMEM supplemented with 10% heat inactivated FBS and cell culture supernatants from cells secreting GM-CSF (for BMDC differentiation) or M-CSF (for BMDM differentiation). 
	NOTE: The amount of cytokines in these supernatants can vary and the working concentration has to be determined empirically. Typically, 2&#8211;3% supernatant from cell lines producing GM-CSF (the recommended starting concentration is 2%) or 5&#8211;10% supernatant from CMG 14&#8211;12 cells producing M-CSF (the recommended starting concentration is 10%) is used. Alternatively, purified commercially available M-CSF at 10 ng/mL and GM-CSF at 20 ng/mL can be used with results virtually identical to cytokine-containing supernatants, i.e., without any effect on the rate of differentiation, infection efficiency and subcellular localization of EGFP-tagged constructs. Antibiotics, including penicillin G (100 IU/mL), streptomycin (100 &#181;g/mL), and gentamicin (40 &#181;g/mL), can be used for cell culture at any step of the protocol unless otherwise stated.</li>
</ol>
2. Production of Retrovirus
CAUTION: Although retroviral vectors are relatively safe when compared to other types of viral vectors, they still pose a potential safety hazard. Therefore, it is crucial to work with the utmost care and appropriate protective equipment, and to adhere to all safety regulations and legal requirements for working with viral particles.
<ol>
	<li>Plate a single cell suspension of Platinum-Eco (Plat-E) packaging cells in a 10 cm Petri dish and cultivate in 15 mL of DMEM containing 10% FBS until 50-60 % confluent (24 h). Cells should grow in a monolayer and should not form clumps in culture.</li>
	<li>Pipette 20 &#181;g of retroviral construct (e.g., the construct expressing the fluorescently tagged protein of interest in the pMSCV vector) and 10 &#181;g of pCL-Eco packaging vector20 into 1 mL of DMEM (without serum and antibiotics) and gently mix.</li>
	<li>To a second tube, add 75 &#181;L of PEI in 1 mL of DMEM (without serum and antibiotics). Incubate 5 min at room temperature (RT) and then mix the contents of both tubes together and incubate for additional 10 min at RT. 
	NOTE: Addition of pCL-Eco is optional. It is coding for the ecotropic viral receptor and may increase the virus titer.</li>
	<li>Carefully replace the medium on Plat-E cells with 8 mL of fresh DMEMsupplemented with 2% FBS. Pre-warm the medium to 37 &#176;C before use. Do not use antibiotics during transfection, since antibiotics may reduce the transfection efficiency.</li>
	<li>Carefully add (in drops) the mixture prepared in Step 2.3 on the Plat-E cells, and incubate for 4 h at 37 &#176;C.</li>
	<li>After the incubation, exchange the medium on Plat-E cells for 10 mL of pre-warmed DMEM containing 10% FBS, and cultivate the cells for 24 h at 37 &#176;C. During this incubation, Plat-E cells will produce virus into the media.</li>
	<li>After 24 h, collect the medium containing retroviral particles from Platinum Eco cells using a 5 mL pipette and transfer it to a 15 mL centrifuge tube (= &#34;supernatant 1&#34; containing ecotropic retroviral particles).</li>
	<li>To avoid contamination by Plat-E cells in viral supernatants, spin the collected virus at 1250 &#215; g for 5 min at 4 &#176;C. For the best results, the virus should be used immediately for infection. 
	NOTE: Aliquots of virus can also be stored at -80 &#176;C for later use. However, it will result in certain reduction in transduction efficiency. Avoid repetitive freezing/thawing of the virus, since it leads to virus degradation.</li>
	<li>Add 10 mL of pre-warmed DMEM with 10% FBS to Plat-E cells and cultivate for another 24 h at 37 &#176;C.</li>
	<li>Repeat steps 2.7. and 2.8. to obtain &#34;supernatant 2&#34;.</li>
</ol>
3. Murine Bone Marrow Cell Isolation
<ol>
	<li>Sacrifice the mouse using cervical dislocation or other approved method. Spray the mouse with 70% ethanol.</li>
	<li>Using tweezers and scissors, remove the skin as well as part of muscles from hind legs. Carefully dislodge the acetabulum from the hip joint without breaking the femur. Cut the paw in the ankle joint. Spray the bones (femur connected to tibia) with 70% ethanol and remove the rest of the muscles using a paper towel.</li>
	<li>Place the bones in a 5 cm Petri dish containing sterile phosphate buffered saline (PBS) with 2% FBS (PBS-FBS).&#160;If preparing more bones, keep the Petri dish on ice until processed.&#160;</li>
	<li>For securing cultivation sterility, perform all the following steps in a tissue culture hood.</li>
	<li>Separate the femur from the tibia without breaking the bone ends (bend in the knee joint and carefully cut with scissors).</li>
	<li>Process the bones one by one. Cut off a very small part of the epiphyses (approximately 1&#8211;2 mm) with scissors while holding the bone in tweezers.</li>
	<li>Use a 30 G needle and a 2 or 5 mL syringe filled with PBS-FBS to flush the bone marrow cells from both ends of the bone into a 15 mL centrifuge tube. Move the needle inside the bone during the flushing in order to remove all the cells. If the needle gets clogged, change it. 
	NOTE: Bones should turn from red to white during flushing. This indicates that the majority of the cells were removed from the bone. Use approximately 2&#8211;3 mL of PBS-FBS per bone.</li>
	<li>Centrifuge the cells at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant and lyse the red blood cells by resuspending the pellet in 2.5 mL of ACK buffer for 2&#8211;3 min at room temperature. During the lysis, filter the bone marrow cells through a 100 &#956;m cell strainer into a fresh 15 mL centrifug tube. Restore the tonicity by adding 12 mL of PBS-FBS. 
	NOTE: Do not exceed 5 min of hypotonic lysis with ACK buffer to avoid cell death.</li>
	<li>Centrifuge immediately at 500 x g for 5 min at 4 &#176;C.</li>
</ol>
4. Bone Marrow Cell Differentiation into Bone Marrow Derived Macrophages
<ol>
	<li>Resuspend the pellet of bone marrow cells in DMEM supplemented with 10 % FBS and antibiotics (see the note after Step 1.4. for antibiotic concentration) and count the cells. For differentiation into BM derived macrophages, plate 5&#8211;10 x 106 of bone marrow cells in a 10 cm non-tissue culture treated (bacterial) Petri dish with 10 mL of pre-prepared DMEM media with serum and M-CSF from Step 1.4. 
	NOTE: The yield of the bone marrow cells is approximately 4 x&#160;107 per 6&#8211;8 week-old C57BL/6J mouse.</li>
	<li>Incubate the cells in cell culture incubator for 3 days at 5% CO2, 37 &#176;C. 
	NOTE: During the first 2 days, cells do not look very vital, as a large number of apoptotic cells is present (cells unable to differentiate into myeloid cells and terminally differentiated cells).</li>
	<li>After 3 days, the bone marrow cell culture begins to look vital and clusters of dividing cells are formed. First adherent cells can already be observed. At this point, supplement the cells with fresh cytokine media.</li>
	<li>Add 10 mL of pre-warmed DMEM media with serum and M-CSF (from Step 1.4) into each 10 cm Petri dish and return it in the cell culture incubator. There is no need to remove the old media during this step. 
	NOTE: Bone marrow macrophages are fully differentiated after 5&#8211;7 days in culture. The best time for harvesting is at day 6&#8211;8, where majority of cells are adherent and the Petri dish is completely covered.</li>
	<li>At day 5, take the Petri dish into the cell culture hood and incline the dish until the media is almost reaching the edge of the dish. Carefully take out 15 mL of the media from the surface near the edge, and the cells tend to stay in the middle of the dish. Add the same volume of pre-warmed media with M-CSF and place the dish back into the incubator. 
	NOTE: If media is aspirated slowly and carefully, almost no cells are lost. However, it is also possible to centrifuge the aspirated media and add the cells back to the culture, to ensure that no non-adherent (i.e., incompletely differentiated) cells are lost.</li>
	<li>For experiments, only adherent cells (macrophages) are used. To harvest cells, on day 6 or 7, remove all media and floating cells. Wash the dish once with pre-warmed PBS without serum.</li>
	<li>Add 5 mL of 0.02% EDTA in PBS, and incubate for 3&#8211;5 min at 37 &#176;C in tissue culture incubator.</li>
	<li>Using a 5 mL pipette, remove the cells from the dish by a stream of PBS-EDTA and place them in a 50 mL centrifuge tube with 25 mL of PBS. If needed, pool more dishes together.</li>
	<li>Centrifuge immediately at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend the macrophage pellet in DMEM media and count the cells. Verify the expression of macrophage surface differentiation markers (CD11b and F4/80) by flow cytometry.</li>
	<li>For experiments requiring the cells to be in suspension, e.g., flow cytometry experiments, qPCR or western blot analysis, use the macrophages directly. For experiments with adherent macrophages, plate the cells in the tissue culture plate according to the experimental setup.</li>
	<li>The cells are already fully differentiated. Keep them in the media suitable for the intended experiment or in the original growth and differentiation media with M-CSF. 
	NOTE: For working with adherent macrophages, transfer them into a new plate at least 6 h before use (ideally overnight) to allow for the full adhesion to the new surface. The small fraction of floating cells can be removed before experiment.</li>
</ol>
5. Bone Marrow Cell Differentiation into Bone Marrow Derived Dendritic Cells
<ol>
	<li>Follow the protocol for BMDMs with adjustments specific for BMDCs described below in steps 5.2&#8211;5.4.</li>
	<li>Resuspend the obtained pellet of bone marrow cells in DMEMsupplemented with 10% FBS and antibiotics and count the cells (by following Step&#160;4.1 of macrophage protocol). For differentiation into dendritic cells, plate 1&#8211;1.5 x 107 bone marrow cells in a 10 cm non-tissue culture treated (bacterial) Petri dish in 10 mL of pre-prepared DMEM media with serum and GM-CSF (from Step 1.4.).</li>
	<li>Follow the same cultivation steps as in BMDM protocol (steps 4.3&#8211;4.5. of macrophage protocol). Use DMEM media with serum and GM-CSF instead of M-CSF. Since for BMDCs the cultivation time is longer (typically 10&#8211;12 days), add 1&#8211;2 additional feedings in 3 day intervals (by removing the supernatant and adding a new cultivation media as described in Step 4.5.).</li>
	<li>This part of the protocol is virtually the same as the corresponding part of the macrophage protocol (Steps 4.6&#8211;4.12. of macrophage protocol). For experiments use only adherent cells. On day 10-12, collect the cells using EDTA, count and plate them on a new surface. Verify the expression of surface differentiation markers of dendritic cells (CD11c+, CD11b+, F4/80-) by flow cytometry.</li>
</ol>
6. Production of BMDMs and BMDCs Expressing EGFP-tagged Protein of Interest
<ol>
	<li>Resuspend the pellet of bone marrow cells obtained in Step 3.10. in DMEM supplemented with 10 % FBS and antibiotics and count the cells. For the infection, use 2&#8211;5 x 106 of BM cells per well of a 6-well tissue culture treated plate.</li>
	<li>Plate the cells in 1 mL of the prepared DMEM media per well, supplemented either with M-CSF for differentiation into BMDMs or with GM-CSF for differentiation in BMDCs. Keep the cells for 4&#8211;6 h in a tissue culture incubator with 5% CO2 at 37 &#176;C.</li>
	<li>Add 2 mL of freshly collected virus (&#34;supernatant 1&#34;) supplemented with polybrene (12 &#181;g/mL, final concentration 8 &#181;g/mL after addition to the cells). 
	NOTE: Frozen aliquot of the virus-containing supernatant can also be used, but efficacy will be lower.</li>
	<li>Centrifuge the plate at 1,250 x g for 90 min at 30 &#176;C (with slow acceleration and deceleration). Then, incubate for 4 h with 5% CO2 at 37 &#176;C.</li>
	<li>Optional: Replace 2 mL of the culture media with fresh medium containing respective cytokine (M-CSF or GM-CSF) and culture with 5% CO2 at 37 &#176;C. On the second day, remove 2 mL of culture media and repeat the whole infection procedure (Step 6.3&#8211;6.4) with 2 mL of freshly collected virus (&#34;supernatant 2&#34;). 
	NOTE: This step may increase infection efficacy. Improvement after the second infection is dependent on the cell type and target protein and in our experience can vary from 30% increase in efficiency to no improvement at all.</li>
	<li>Collect the non-adherent cells, transfer to a 15 mL centrifuge tube, and spin at 500 x g for 5 min (4 &#176;C). Discard the supernatant.</li>
	<li>Resuspend the cell pellet in 10 mL of culture media with M-CSF or GM-CSF, place the cells into a 10 cm non-tissue culture treated Petri dish and culture at 37 &#176;C, 5% CO2. Optimal number of cells for a 10 cm dish is 5&#8211;10 x 106 for BM-derived macrophages and 10&#8211;15 x 106 for BM-derived dendritic cell. 
	NOTE: Smaller dishes or plates can be used, but cell numbers must be adjusted accordingly.</li>
	<li>Follow the macrophage and dendritic cell cultivation and differentiation protocol described in step 4 and 5.</li>
</ol>

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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 authors have nothing to disclose.

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>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Lachema, Brno, Czech Republic</td><td>N/A</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</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&amp;Aacute; SOL&#039; 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&amp;aacute;lov&amp;eacute;, 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 &amp;micro;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 ...

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JoVE Journal

Biology

10.3K Views.  Institute of Molecular Genetics of the ASCR. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van macrofaag en dendritische celbiologie over de subcellulaire lokalisatie en dynamiek van verschillende eiwitten in deze celtypen. De belangrijkste voordelen van deze techniek zijn dat het relatief eenvoudig, goedkoop, en toont een betere efficiëntie en stabiliteit van de expressie dan de meerderheid van de andere beschikbare methoden. Het aantonen van de procedure zal Jarmila Kralova zijn, een student uit mi...

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

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

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

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

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Immunology and Infection

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% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 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% ...

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 ...

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.

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. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

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 ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

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 ...

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

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