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

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

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

<ol>
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K., Costanzi, E., Haas, M., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pCL+vector+system:+rapid+production+of+helper-free,+high-titer,+recombinant+retroviruses.">The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.</a> Journal of Virology. 70 (8), 5701-5705 (1996).</li><li>Janssen, E., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptor+proteins+in+lymphocyte+activation.">Adaptor proteins in lymphocyte activation.</a> Current Opinion in Immunology. 15 (3), 269-276 (2003).</li><li>Ferguson, P. J., Laxer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+discoveries+in+CRMO:+IL-1beta,+the+neutrophil,+and+the+microbiome+implicated+in+disease+pathogenesis+in+Pstpip2-deficient+mice.">New discoveries in CRMO: IL-1beta, the neutrophil, and the microbiome implicated in disease pathogenesis in Pstpip2-deficient mice.</a> Seminars in Immunopathology. 37 (4), 407-412 (2015).</li><li>Holleman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+outcome+predictor+in+acute+leukemia+1+(OPAL1)+gene+is+not+an+independent+prognostic+factor+in+patients+treated+according+to+COALL+or+St+Jude+protocols.">Expression of the outcome predictor in acute leukemia 1 (OPAL1) gene is not an independent prognostic factor in patients treated according to COALL or St Jude protocols.</a> Blood. 108 (6), 1984-1990 (1984).</li><li>Zjablovskaja, P., Danek, P., Kardosova, M., Alberich-Jorda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+Differentiation+of+Murine+Myeloid+Precursor+32D/G-CSF-R+Cells.">Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells.</a> Journal of Visualized Experiments: JoVE. (132), (2018).</li><li>Kralova, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Transmembrane+Adaptor+Protein+SCIMP+Facilitates+Sustained+Dectin-1+Signaling+in+Dendritic+Cells.">The Transmembrane Adaptor Protein SCIMP Facilitates Sustained Dectin-1 Signaling in Dendritic Cells.</a> Journal of Biological Chemistry. 291 (32), 16530-16540 (2016).</li><li>Maess, M. B., Wittig, B., Lorkowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+of+human+THP-1+macrophages+by+nucleofection.">Highly efficient transfection of human THP-1 macrophages by nucleofection.</a> Journal of Visualized Experiments: JoVE. (91), e51960 (2014).</li><li>Bowles, R., Patil, S., Pincas, H., Sealfon, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocol+for+efficient+transfection+of+dendritic+cells+without+cell+maturation.">Optimized protocol for efficient transfection of dendritic cells without cell maturation.</a> Journal of Visualized Experiments: JoVE. (53), e2766 (2011).</li><li>Siegert, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+siRNA+into+mouse+bone+marrow-derived+macrophages+and+dendritic+cells.">Electroporation of siRNA into mouse bone marrow-derived macrophages and dendritic cells.</a> Methods in Molecular Biology. 1121, 111-119 (2014).</li><li>Lee, C. 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The expression of protein of interest in target cells is a key step in many types of biological studies. Differentiated macrophages and dendritic cells are difficult to transfect by standard transfection and retroviral transduction techniques. Bypassing the transfection of these differentiated cells with retroviral transduction of bone marrow progenitors, followed by differentiation when they already carry the desired construct, is a critical step allowing the expression of ectopic cDNAs in these cell types. An example o...

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

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

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

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

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

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

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

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

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

This method can help answer key questions in the fields of macrophage and dendritic cell biology about the sub-cellular localization and dynamics of various proteins in these cell types. The main advantages of this technique are that it is relatively simple, inexpensive, and demonstrates a better efficiency and stability of expression than the majority of other available methods. Demonstrating the procedure will be Jarmila Kralova, a grad student from my laboratory.To produce the retrovirus, plate a single-cell suspension of Platinum Eco-packaging cells in a 10 centimeter petri dish and cultivate the cells in 15 milliliters of DMEM, supplemented with 10%fetal bovine serum, or FBS, until the culture is 50 to 60%confluent. The next day, add 20 micrograms of the retroviral construct of interest and 10 micrograms of PCL eco-packaging vector to 1 milliliter of DMEM without serum, with gentle mixing. Add 75 microliters of PEI in 1 milliliter of DMEM without serum and antibiotics to a second tube for a five minute incubation at room temperature, and mix the contents of both tubes together for an additional 10 minute incubation at room temperature.Next, carefully replace the medium on the Platinum Eco cells with 8 milliliters of fresh 37 degree Celsius DMEM supplemented with 2%FBS and carefully add the transfection mixture in drops to the plate. After a 4 hour incubation at 37 degrees Celsius, replace the supernatent with 10 milliliters of 37 degree Celsius DMEM, supplemented with 10%FBS for a 24 hour incubation at 37 degrees Celsius. The next day, use a 5 milliliter pipette to transfer the ecotropic retroviral particle-containing supernatent to a 15 milliliter centrifuge tube and remove the debris and containing cells by centrifugation.Meanwhile, add 10 milliliters of 37 degrees Celsius DMEM plus 10%FBS to the culture dish for another 24 hour incubation at 37 degrees Celsius and collect a second round of viral particles as just demonstrated. To harvest the bone marrow, in a tissue culture hood, use tweezers and scissors to remove the skin and part of the muscles from the hind limbs and carefully dislodge the acetabulum from the hip joint without breaking the femur. Cut the paws at the ankle joints and spray the bones with 70%ethanol.Then remove the rest of the muscle with a paper towel and place the bones in a 5 centimeter petri dish of PBS supplemented with 2%FBS. Next, bend the knee joint of each bone set and carefully separate the bones with scissors. Use the scissors to remove about one to two millimeters of each epiphysis of one bone and use a two or five milliliter syringe equipped with a 30 gauge needle loaded with fresh PBS plus FBS to flush the bone marrow from both ends of the bone into a 15 milliliters centrifuge tube.When the marrow has been collected from each of the bones, pellet the bone cells by centrifugation and re-suspend the pellet in 2.5 milliliters of red blood cell lysis buffer for two to three minutes at room temperature. After lysing, filter the cells through a 100 micrometer cell strainer into a new 15 milliliter centrifuge tube and restore the tenacity with 12 milliliters of PBS plus FBS. Re-suspend the pellet in DMEM, supplemented with 10%FBS and antibiotics for counting and plate five to 10 times 10 to the sixth bone marrow cells in a 10 centimeter, non-tissue culture treated petri dish in 10 milliliters of DMEM supplemented with serum and macrophage colony stimulating factor, or MCSF.On day five of culture, carefully tilt the cell culture dish and remove all but approximately 5 milliliters of medium from the surface near the edge of the dish. Then add 20 milliliters of fresh 37 degree Celsius medium supplemented with FBS and MCSF to the dish and return the cells to the cell culture incubator. To harvest the cells, on day six to seven, remove all of the medium and floating cells and wash the culture with 37 degree Celsius PBS.Detach the adherent cells with five milliliters of 0.02%EDTA and PBS for three to five minutes at 37 degrees Celsius in the tissue culture incubator and use a five milliliter pipette to gently flush the dissociated cells from the plate bottom. Then transfer the cells to a 50 milliliter centrifuge tube containing 25 milliliters of fresh PBS. To induce EGFP tagged protein expression in bone marrow derived antigen presenting cells, at the beginning of the cell differentiation protocol, dilute the bone marrow cells to a two to five times 10 to the sixth cells per milliliters of DMEM plus FBS and antibiotics conentration and plate the cells of one milliliter of medium per well, supplemented with the appropriate differentiation cytokine.After four to six hours in the cell culture incubator, add two milliliters of the first round of collected virus supplemented with polybrene and transduce the cell cultures by centrifugation and a four hour incubation in the cell culture incubator. At the end of the transduction period, transfer the nonadherent cells from each well into individual 15 milliliter centrifuge tubes and collect the cells by centrifugation. If larger amount of cells is needed, the cells can be infected with the same constract in multiple wells and pooled before differentiation.Then re-suspend the pellets in 10 milliliters of culture medium supplemented with the appropriate differentiation cytokine and plate the cells onto one 10 centimeter non tissue culture treated petri dish per tube, for their six to eight day culture, as just demonstrated. Evaluation of the efficacy of Platinum Eco transfection by flow cytometry after the collection of the second virus containing supernatent reveals a mean transfection efficiency of 62%for PSTPIP2-EGFP and 53%for OPAL1-EGFP. Retroviral transduction does not affect the differentiation status of the bone marrow derived cells with more than 90%of the cells in both cultures demonstrating positivity for their respective markers after transduction with either virus.And with the appropriate, characteristic morphological changes observed for both types of differentiated cell. Of note, the overall expression of EGFP was lower in bone marrow derived dendritic cell cultures compared to bone marrow derived macrophage EGFP expression, regardless of type of retrovirus. While attempting this procedure, it is important to remember to use non tissue, culture treated plastic dishes that are typically used for cultivating bacteria if you plan to remove cells from the dish for experiment.Following this procedure, other methods live cell imaging, super resolution microscopy or other types of microscopy can be employed to answer additional questions about the sub-cellular localization and dynamics of dendritic cell and macrophage proteins of interest. This technique paved the way for researchers in the immunology field in exploring the distribution and spacial relationships of proteins expressed by macrophages and dendritic cells and in furthering our understanding of the function of these protein during immune responses. Don't forget that working with viral particles can be extremely hazardous and that precautions, such as minimalizing aerosol generation, wearing protective equipment, and using a laminar flow hood, should always be taken while performing this procedure.

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

Research

JoVE Journal

Biology

Culture of myeloid dendritic cells from bone marrow precursors

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

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

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.