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 of successful use of this method can be found in our recent publication25. Here, we provide a cost-effective protocol for achieving stable expression of the construct of choice in bone marrow-derived dendritic cells and macrophages using this approach. The procedure we present is relatively inexpensive and simple, yet delivering very good results. Reagents used in this protocol allow for its routine use even under a relatively restrictive budget. The protocol for Plat-E transfection employs PEI as a transfection reagent. Compared to other chemical transfection agents, PEI is of a very low cost, while its efficiency is similar to the majority of other widely used compounds. However, PEI can be replaced with many different commercially available transfection reagents in this step without any loss of efficiency. As a guiding principle, transfection protocols known to work with commonly used HEK293 cells typically perform well with Plat-E cells, too. Another cost-effective measure is the utilization of cytokine-containing supernatants instead of purified recombinant cytokines. The use of these supernatants requires some optimization. However, when the standard protocol for their preparation is established and followed, the variability between individual batches of these supernatants becomes very low, usually requiring no changes in working concentrations between individual lots. The efficiencies of BMDM and BMDC differentiation with these supernatants are, in our experience, identical to purified cytokines.
In addition to retroviral transduction, other well-established methods of mammalian cell transfection exist, including chemical transfection (typically using cationic lipids or cationic polymers forming complexes with DNA), electroporation and the use of other types of viral vectors, mainly adenoviral and lentiviral systems26,27,28,29,30. Although very high titers and efficiencies can be achieved with adenovirus-based gene delivery, the preparation of corresponding plasmids and viral particles are more difficult and time-consuming than in the case of retroviral systems29. In addition, adenovirus elicits inflammatory response in dendritic cells and macrophages29,31,32 and for optimum performance in murine hematopoietic cells mouse strain carrying transgenic adenovirus receptor is required33. On the other hand, the generation of lentiviral particles carrying the gene of interest is a relatively simple process, virtually identical to the one utilized for retroviruses. In contrast to the ecotropic retroviral vectors used in this protocol, lentiviruses are capable of infecting non-proliferating cells of multiple species, including humans34. This may be an important advantage under specific experimental conditions. However, this feature also greatly compromises the safety of these vectors. In our opinion, for gene delivery to BMDMs and BMDCs, retroviruses provide the best balance of efficiency, safety and ease of use. Retroviral DNA constructs can be easily prepared using simple standard molecular cloning techniques. Virus is produced by packaging the cell lines directly to the culture supernatant and further virus purification is usually not necessary. Ecotropic retroviruses also do not readily infect human cells, which makes their use relatively safe. However, there also are some general disadvantages associated with the use of retroviral vectors. The major limiting factor is that these viruses infect only proliferating cells35. This feature does not significantly affect the protocol described here, but it limits the range of applications where retroviral vectors can be used. The size of the gene of interest that can be cloned into these vectors is also limited and the viral particle titer decreases with increasing insert size. With pMSCV vectors, we usually start seeing effects of insert size at around 3 kbp. With further increases in insert size, the infection efficiency gradually declines.
The chemical transfection and electroporation are easier to use, safer and less time-consuming than any of the virus-based procedures26,27,28,30. However, in BMDMs and BMDCs, they can stimulate responses to foreign nucleic acids31 and they generate more cellular stress. In addition, some chemical transfection reagents increase cell auto-fluorescence that may interfere with flow cytometry or microscopy analyzes. Due to the transient nature of expression, they can only be used with mature differentiated BMDMs or BMDCs. In contrast, the sequences introduced with retroviral vectors are permanently integrated into the genome of the target cells and allow for a stable long-lasting expression compatible with the time scale of the differentiation protocols34,35. However, this feature also increases the risk of insertional mutagenesis. Due to the relatively random nature of the vector integration, its effects on large populations of cells are limited. However, they may be visible at the level of individual cells. Additional problems may arise when a construct expressed from the retroviral vector affects dendritic cell or macrophage differentiation, resulting in failure to generate differentiated BMDMs or BMDCs from the infected progenitors. To some extent, this may be overcome by adjusting the infection conditions to achieve low expression levels (e.g., by reducing the virus titer) or through the use of an inducible expression system. The use of EGFP fused to the protein of interest or as a reporter also allows for sorting of cells with expression level corresponding to experiment goals and limitations. Finally, we should also mention problems common to all transfection/transduction procedures. These include mainly overexpression artefacts, such as protein misfolding, mislocalization and toxicity36. Protein toxicity could be the reason why OPAL1 was relatively difficult to express. Its example clearly illustrates that the nature of the expressed protein can substantially affect the effectiveness of this method. However, despite this, we were able to obtain sufficient quantities of OPAL1-EGFP expressing cells for microscopy analysis with this method, demonstrating its usefulness even when dealing with difficult targets. In addition, it would be possible to increase the percentages of transduced cells by FACS sorting if required by a particular application. The low infection efficiency can also be partially overcome by increasing viral particle concentration using various methods or ready-to-use kits. In our hands, ultrafiltration of the viral supernatant on centrifugal filters with a molecular weight cut off of 100 kDa has proven to provide the best balance between efficiency and required effort.
Bone marrow derived macrophages and dendritic cells are widely used tools in phagocyte immunology. They are more physiologically relevant than available cell lines. They can be generated in relatively high numbers and, at the same time, lack the genetic heterogeneity and instability characteristic of cell lines. Another advantage is that they can be generated from genetically modified mice to study the effects of genetic modification on a relatively abundant and homogenous cell population. This is particularly useful in biochemical studies, where relatively large amounts of cells are typically required. The ability to transduce these cells with cDNA constructs opens up additional possibilities of research based on the reconstitution or complementation of genetic defects in these cells and structure-function analysis.

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 inflammatory response22. It is a cytoplasmic protein which can also be recruited to cellular membranes via its F-bar domain. Second protein is a transmembrane adaptor OPAL1, expected to be associated with cellular membranes. Its physiological function is still unknown. However, in acute lymphoblastic leukemia, expression of OPAL1 is associated with better prognosis23.
cDNA constructs coding for PSTPIP2 or OPAL1 fused via a short linker (GSGGGS or Myc-tag, respectively) to EGFP at the C-terminus were cloned into the pMSCV retroviral vector using standard methods of cDNA cloning. This construct was then transfected into Plat-E cells together with the packaging vector pCL-Eco. The resulting supernatants containing retroviruses were used for the transduction of bone marrow cells, followed by the differentiation into BMDMs and BMDCs. The efficacy of Plat-E transfection was evaluated by flow cytometry after the collection of the second virus-containing supernatant. Mean transfection efficiency was 62% for PSTPIP2-EGFP and 53% for OPAL1 and the results were highly reproducible (Figure 1A, B). OPAL1 construct seemed to be more toxic for Plat-E cells (assessed by the appearance of floating/dying cells in culture), resulting in a reduction in the percentages of transfected cells.
Differentiation status of the bone marrow derived macrophages and dendritic cells (transduced with PSTPIP2-EGFP and OPAL1-EGFP retroviral constructs) was assessed by flow cytometry. Mature macrophage population is defined by CD11b and F4/80 expression, while dendritic cells express the CD11c lineage marker. More than 90% of cells in both types of culture were positive for their respective markers (Figure 2A, B). Finally, we determined the expression level of PSTPIP2-EGFP and OPAL1-EGFP constructs in BMDMs and BMDCs by a simple flow cytometry measurement of EGFP fluorescence. The mean percentage of EGFP-positive macrophages was 71% for PSTPIP2-EGFP and 62% for OPAL1-EGFP (Figure 3A). In case of dendritic cells, the efficiency was lower, 32% for PSTPIP2 and 9% for OPAL1 (Figure 3B). The results of multiple experiments demonstrate the reproducibility of this method (Figure 3C).
We typically do not determine the virus concentration in the supernatants that we use in infections. We prefer to use the virus supernatants fresh, immediately after collection, while the virus titer determination requires three additional days. As a result, the information on virus titer can only be obtained ex post. However, it can still be useful when addressing technical issues and problems. To assess the virus concentration in supernatants from Plat E cells transfected with PSTPIP2-EGFP and OPAL1-EGFP constructs, we incubated NIH-3T3 cells with serially diluted virus-containing supernatants collected from these transfected Plat-E cells and determined virus titer exactly as described by Zjablovskaja et al in previously published JoVE article24. In three independent experiments, the virus titer ranged from 1.1 x 106 to 4.4 x 106 TU/mL. We did not observe any substantial differences between PSTPIP1-EGFP and OPAL1-EGFP constructs and between supernatants from day 1 and day 2. When these supernatants were used for bone marrow cell infections according to the protocol we are describing in this article, the multiplicity of infection (MOI) ranged from 1.1 to 4.4. Interestingly, within this range, we did not observe any correlation between MOI and infection efficiency.
In Figure 4, PSTPIP2-EGFP and OPAL1-EGFP expressed in BMDMs and BMDCs were visualized by confocal microscopy. Fully differentiated macrophages and dendritic cells have a characteristic shape. The change in morphology from small rounded progenitor cells to the large cells of irregular shapes confirms successful differentiation. In macrophages, PSTPIP2 was cytoplasmic with partial localization at the plasma membrane. OPAL1 appeared to be also partially targeted to the plasma membrane. The rest was likely associated with intracellular membranes, such as the endoplasmic reticulum and Golgi complex. However, to confirm this localization, specific organelle markers would have to be used. In dendritic cells, the membrane localization was less apparent.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58081/58081fig1.jpg" /> 
Figure 1: Efficiency of Plat-E cell transfection. For transfection of Plat-E cells, two constructs encoding adaptor proteins PSTPIP2 and OPAL1 fused with EGFP (PSTPIP2-EGFP and OPAL1-EGFP) were cloned into the pMSCV vector. Standard PEI transfection was performed. The efficacy of transfection was evaluated by flow cytometry of the Plat-E cells after the collection of the second viral supernatant. (A). Representative flow cytometry plot. (B). Graph showing results of four independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58081/58081fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58081/58081fig2.jpg" /> 
Figure 2: Assessment of the differentiation status of BMDMs (A) and BMDCs (B). Surface expression of specific macrophage and dendritic cell lineage markers was measured by flow cytometry at day 8 of cultivation. Dead cells were gated out based on their side and forward scatter properties and staining with Hoechst 33258. The results are representative of at least 3 independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58081/58081fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58081/58081fig3.jpg" /> 
Figure 3: Assessment of the expression of PSTPIP2-EGFP and OPAL1-EGFP. EGFP fluorescence in BMDMs (A) and BMDCs (B) retrovirally transduced with PSTPIP2-EGFP and OPAL1-EGFP constructs was measured by flow cytometry at day 8 of cultivation. (C). Graph showing the results of multiple independent experiments. BMDMs and BMDCs were gated as in Figure 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58081/58081fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58081/58081fig4.jpg" /> 
Figure 4: Representative images of macrophages and dendritic cells expressing PSTPIP2 or OPAL1. BMDMs (A) and BMDCs (B) expressing PSTPIP2 and OPAL1 were visualized by live imaging confocal microscopy. EGFP fluorescence in green is shown on the left side of each panel, bright field image on the right. Scale bar = 10 &#181;m. The results are representative of at least three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58081/58081fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

expression-fluorescent-fusion-proteins-murine-bone-marrow-derived

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K Views.  Institute of Molecular Genetics of the ASCR. 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.

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

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 infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

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% 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% ...

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 MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

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 (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

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 in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

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 to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

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 fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

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

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 the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

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

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...