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-CSF alone5,6, GM-CSF and IL-47, or GM-CSF and FLT3 ligand8,9 generate BMDCs more closely resembling inflammatory DCs (CD11chigh, MHCIIhigh CD11b+)10. While these in vitro cultures are used to generate macrophages or DCs, it is unclear if each culture gives rise to pure populations. For example, although adherent cells in the GM-CSF cultures are described to be macrophages5, the non-adherent cells from the same culture are used as DCs6,11-13, with the presumption that they are homogeneous and any observed variability is due to different stages of development14,15. Furthermore, studies have found GM-CSF to be an essential growth factor for alveolar macrophage development in vivo16,17, and can be used in vitro to generate alveolar-like macrophages16,17,18.
Other than adherence, the procedures for generating macrophages and DCs from GM-CSF treated bone marrow cultures are very similar suggesting heterogeneity may exist within GM-CSF bone marrow cultures. This indeed seems to be the case as two papers report the presence of BMDMs in the non-adherent fraction of BMDC cultures. In one paper, they identified a population of cells as CD11c+, CD11b+, MHCIImid, MerTK+, and CD115+, which expressed a gene expression signature that most closely resembled alveolar macrophages and had a reduced ability to activate T cells19. The second paper used MHCII and FL-HA to identify an alveolar macrophage-like population (CD11c+, MHCIImid/low, FL-HAhigh) that was distinct from immature (CD11c+, MHCIImid, FL-HAlow) and mature DCs (CD11c+, MHCIIhigh), both phenotypically and functionally18. These papers both illustrate that GM-CSF BMDC cultures are heterogeneous, containing both macrophage and DC populations indicating that care should be taken when interpreting data from BMDC cultures.
This protocol describes how to isolate bone marrow, culture bone marrow cells in GM-CSF, and identify the alveolar macrophage-like population from the immature and mature DCs in the bone marrow culture by flow cytometry using FL-HA binding and MHCII expression. This procedure is based on the established procedure of Lutz et al.6 and is able to generate 5 – 10 x 106 non-adherent cells on day 7 of a 10 ml culture. The culture is usable from days 7 to 10 and yields a heterogeneous population of macrophages, immature and mature DCs, as well as some progenitors at day 7. This provides a simple method to grow and isolate in vitro alveolar-like macrophages in large quantities.
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…
The authors have nothing to disclose.
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.
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 |