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Biology

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

Published: October 30th, 2018

DOI:

10.3791/58081

1Laboratory of Leukocyte Signalling, Institute of Molecular Genetics of the ASCR, 2Faculty of Science, Charles University, 3Department of Biophysical Chemistry, J. Heyrovsky Institute of Physical Chemistry 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.

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.

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 ....

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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

  1. 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 µL of 0.5 M ethylenediaminetetraacetic acid (EDTA) and filter-sterilize.
  2. Prepare polye.......

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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.......

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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.......

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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).

....

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

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