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Immunology and Infection

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Published: June 25th, 2016

DOI:

10.3791/54194

1Microbiology and Immunology, University of British Columbia

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Several in vitro culture methods have been described to generate bone marrow-derived macrophages (BMDMs) and bone marrow-derived DCs (BMDCs) using one or a combination of growth factors. BMDMs can be generated by culturing bone marrow cells using either macrophage colony stimulating factor (M-CSF) or GM-CSF1,2. For BMDCs, the addition of FLT3 ligand to the bone marrow culture gives rise to non-adherent classical and plasmacytoid DCs (CD11chigh/MHCIIhigh and CD11clo, B220+ respectively) after 9 days in culture3,4. In contrast, non-adherent cells generated after 7 to 10 days in culture with GM-CS....

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Mice were euthanized in accordance with the Canadian Council on Animal Care guidelines for ethical animal research by procedures approved by the University of British Columbia Animal Care Committee.

1. Acquiring a Single Cell Bone Marrow Suspension from Mouse Femur and Tibia

  1. Switch on a biological safety cabinet (BSC) 15 min prior to start of procedure to purge cabinet air and allow for air flow stabilization, then clean/spray BSC surface with 70% ethanol. Keep everything as sterile as possible for the.......

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A flowchart summarizing the major steps of this method is shown in Figure 1. The density and morphology of the bone marrow culture at different times of culture are shown in Figure 2. At day 1, the cells are small and sparse but by day 3, there are more cells, some are larger and a few have begun to adhere. By day 6 there is a definite adherent and non-adherent fraction (Figure 2A). The culture can be harvested from day 7 - 10 with a high.......

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In this manuscript, we provide a method for generating GM-CSF derived macrophages and DCs from a single mouse bone marrow culture that is adapted from Lutz et al.6. MHCII  expression and FL-HA binding distinguishes between immature DCs and macrophages in this culture (see Figure 3C), which previously has been difficult. This, together with another report by Helft et al.19, demonstrates heterogeneity within GM-CSF induced BMDC cultures that were previously thou.......

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This work was funded by the Canadian Institutes of Health Research (CIHR) (Grant MOP-119503) and the Natural Sciences and Engineering Council of Canada (NSERC). NSERC also supported summer studentships to Y.D. and A.A. YD is supported by the University of British Columbia (UBC) with a 4-year fellowship award, A.A is supported by CIHR with a graduate student Master's award (CGS-M). We thank Calvin Roskelley for assistance with the microscope used to generate the images in Figure 2. We also acknowledge support from the UBC Animal and Flow Cytometry Facilities.

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Name Company Catalog Number Comments
Flow Cytometer BD  LSR-II
Automated Inverted Microscope  Leica  DMI4000 B
Centrifuge  Thermo Fisher ST-40R
Biosafety Cabinet Nuaire NU-425-600
Syringe 1 ml BD 309659
26 1/2 Gauge Needle BD 305111
50 ml Conical Tube  Corning 357070 *Falcon brand
Eppendorf tubes (1.5 ml) Corning MCT-150-C
5 ml polystyrene round bottomed tubes Corning 352052
Dissection Tools Fine Science Tools  *Various  *Dissection scissors, dumont forcep and standard forcep 
Hemocytometer  Richert 1490
Sterile 100 x 15 mm Petri Dish Corning 351029 *Falcon brand
2-Mercaptoethanol Thermo Fisher 21985-023
Ammonium Chloride BDH BDH0208-500G
Bovine Serum Albumin Fisher Bioreagents BP1600-1
Brefeldin A Sigma B7651-5MG
EDTA Sigma E5134-1KG Ethylenediaminetetraacetic acid
Fetal Bovine Serum Thermo Fisher 16000-044
Hank's Balanced Salt Solution Thermo Fisher 14175-095 
HEPES Thermo Fisher 15630-080 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
L-Glutamine Sigma G8540-100G
LPS Ultrapure Invivogen tlrl-3pelps
MEM Non-Essential Amino Acids Solution  Thermo Fisher 11140-050
Penicillin/Streptomycin 100x Thermo Fisher 15140-122
Potassium Phosphate Monobasic BDH BDH0268-500G
Potassium Chloride BDH BDH9258-500G
Recombinant GM-CSF Peprotech 315-03-A
Rooster Comb Sodium Hyaluronate  Sigma H5388-1G *Used to make fluoresceinated hyaluronan
RPMI-1640  Thermo Fisher 21870-076 No sodium pyruvate no glutamine. Warm media to 37oC before using. 
Sodium Chloride Fisher  5271-10  
Sodium Phosphate Dibasic Sigma 50876-1Kg
Sodium Pyruvate Sigma P5290-100G
Tris(hydroxymethyl)aminomethane Fisher Bioreagents BP152-5
Trypan Blue Sigma T8154
Anti-Fc Receptor (unlabeled), Tissue Culture Supernatant N/A N/A Clone: 2.4G2
Anti-CD11c PeCy7 eBioscience 25-0114-82 Clone: N418
Anti-Gr-1 efluor450 eBioscience 48-5931-82 Clone: RB6-8C5
Anti-MHCII APC eBioscience 17-5321-82 Clone: M5/114.15.2
Biotinylated Anti-MerTK Abcam BAF591 Goat polyclonal IgG
Streptavidin PE eBioscience 12-4317-87
Propidium Iodide Sigma P4170-25MG
DAPI (4',6-diamidino-2-phenylindole) Sigma D9542-5MG

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