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.
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
2. Plating and Culturing of Bone Marrow Cells (BMCs)
3. Collecting and Preparing BMDC and BMDMs for Flow Cytometry
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 higher percentage of CD11c+ cells present at day 10. Figure 2B shows the day 7 culture before harvest, as well as the separated adherent and non-adherent cell fractions. The adherent fraction consists of the cells left after the light washes that remove the non-adherent fraction. The non-adherent fraction is greatly enriched for cells with a dendritic morphology, which can be seen more clearly in the digitally magnified images in the insets in Figure 2B. Once the non-adherent fraction is removed (day 7 or day 10), the cells are analyzed by flow cytometry after labeling with fluorescent antibodies against key cell surface markers, as described above. Figure 3 shows representative flow cytometry plots of the non-adherent fraction of the culture at day 7 and day 10.
For the identification of both macrophages and DCs, gate on CD11c+ and Gr1– cells after first gating on size and live cells (Figure 3A and B). In day 7 cultures, the CD11c+ Gr1– population in the non-adherent cell fraction is typically 60 to 70% of total live cells, and this is increased to 90% or more on day 10 (Figure 3B). The CD11c+ Gr1– population at day 7 and day 10 can be divided into three main subsets using MHCII and FL-HA binding: MHCIImid/low FL-HA bindinghigh macrophages (P1), MHCIImid FL-HA bindinglow (P2) cells containing immature DCs, and MHCIIhigh FL-HA bindinglow mature DCs (P3) (Figure 3C and reference18). The FL-HAlow, MHCIIlow population (P0) observed at day 7 has been shown to contain progenitors for the P1-P3 cells18. This population is not evident at day 10 implying that further maturation of the culture has occurred. This is also supported by the greater percentage of CD11c cells in the culture as well as slightly higher percentages of the P2 and P3 DC populations at day 10. Figure 3D shows MerTK, considered to be a macrophage marker, is highly expressed on both macrophage and immature DC populations (P1 and P2), but is expressed to a lower extent on mature DCs (P3 cells). Notably, analysis of the adherent fraction reveals the presence of the P0, P1 and P2 populations, but not the P3 mature DC population (data not shown). Thus macrophages are present in the adherent and non-adherent fractions but only mature DCs are present in the non-adherent fraction.
Figure 1: A Flow Chart Indicating the Key Steps of the Method. Primary cells are harvested from the bone marrow, plated and maintained in culture for 7 – 10 days when they are harvested for use in further experiments, purified or used in FACS analysis. Please click here to view a larger version of this figure.
Figure 2: Microscopic Examination of GM-CSF Derived Bone Marrow Cells. A) Images of the bone marrow culture at day 1, 3, and 6 showing the increase in cell number, changes in morphology, and emergence of distinct adherent and non-adherent fractions. B) Left panel shows the day 7 bone marrow culture and the typical cell density. The middle panel shows the day 7 adherent cells after harvest of the non-adherent fraction and the right panel shows the non-adherent fraction harvested at day 7. Insets are digitally magnified images of cells in this fraction with dendritic morphology. The white scale bar represents 50 µm. Please click here to view a larger version of this figure.
Figure 3: Representative Phenotype of the GM-CSF Culture at Day 7 and Day 10. Non-adherent cells from the culture are labeled with CD11c, Gr1, MHCII, FL-HA, and MerTK at day 7 or day 10 and analyzed for their phenotype by flow cytometry. A) Cells were first gated on size then live cells. B) Shows a plot of the gated cells with CD11c and Gr1 (the neutrophil/monocyte marker) and identifies the CD11c+Gr1– population containing the macrophage and dendritic cells. Gating on this population, C) shows the flow cytometry plot of MHCII and FL-HA binding, which shows the separation of the alveolar-like macrophages (P1) from immature DCs (P2) and mature DCs (P3), as described in Poon et al. 18. D) Histograms comparing MerTK expression between the P1 (macrophages), P2 (immature DCs), and P3 populations (mature DCs) at day 7 and day 10. Please click here to view a larger version of this figure.
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 thought to be only DCs. Helft et al.19 used quite different culture conditions to generate BMDCs yet also found a significant macrophage population. GM-CSF induced macrophages resemble alveolar macrophages and GM-CSF induced DCs resemble inflammatory DCs, whereas FLT3 ligand supplemented BMDC cultures produce classical and plasmacytoid DCs3,4. Different DC and macrophage populations are also observed in vivo under homeostatic and inflammatory conditions, and there is much discussion in the field as to what defines a DC and a macrophage10. This is particularly true when it comes to monocyte derived inflammatory DCs and this is also relevant in this BMC. It is thus important to use several phenotypic markers, functional data, and possibly gene expression data to characterize a particular population. It is also possible that these in vitro DC and macrophage populations may be further sub-divided as more functional and phenotypic markers are discovered.
In this method, the markers CD11c, Gr1, MHCII, and FL-HA are used to distinguish macrophages from immature and mature dendritic cells in the non-adherent fraction of the BMC culture. This is different to Helft et al.19, who used the markers CD11c, CD11b, CD115, CD135, MerTK, and MHCII to distinguish a macrophage population from mature DCs. MerTK expression levels could distinguish between macrophages (P1) and mature DCs (P3), but not immature DCs (P2) (Figure 3D). Thus FL-HA and MHCII are useful markers to discriminate macrophages from both immature and mature DC populations. These macrophages are also functionally distinct from DCs in this BMC culture and this is demonstrated in more detail elsewhere18. Briefly, after LPS stimulation the BMC macrophages produce different cytokines than mature DCs and do not activate naïve T cells18. These macrophages also closely resemble alveolar macrophages, both phenotypically and functionally as both bind FL-HA and express CD11c, MerTK, CD200R, CD206 and F4/80 and after LPS stimulation produce TNF-α yet are unable to activate naïve T cells18. However, there are also differences: the BMC macrophages are CD11b+ and have lower levels of Siglec F compared to ex-vivo alveolar macrophages, suggesting these cells are similar but not identical to alveolar macrophages.
There are several important steps to ensure the success of this protocol. It is important to maintain sterility from exposing the bone marrow onwards. When moving in and out of the BSC, spray gloved hands and objects with 70% ethanol. Although it is helpful to rinse tools in 70% ethanol to sterilize, ethanol is toxic to bone marrow cells, so air-dry the tools and ensure the ethanol has evaporated before bringing bones or cells in contact with them. In addition, avoid contacting mouse tissues or cells with non-sterile equipment or hands during the bone marrow harvest. Although penicillin and streptomycin supplementation is provided in the media, careless handling of the cells may lead to yeast or fungal contamination. In addition, since immune cells with phagocytic activity are generated in this culture, minor contamination may not be detected by simply observing the cell culture microscopically, thus is it important to identify the signs of contamination. For example, cell numbers may be reduced and the expression of cell activation markers such as CD40 and CD86 may be increased when analyzed by flow cytometry. Media color may also turn yellow reflecting a pH change due to contamination. When contamination occurs, the BMC culture is unusable. To maintain experimental consistency, it is important to generate a single cell suspension from the bone marrow by vortexing and obtain an accurate cell count to set up the BMC cultures. Also, if any connective tissue from the bone ends up in the single cell suspension, filter through a sterile 70 micron filter to remove it. Typical cell yields for one plate of the non-adherent fraction at day 7 are between 5 – 10 x 106 cells. Cell numbers are usually a good indication that the protocol is working well. With this procedure we usually obtain between 2 – 5 x 106 cells on day 10, whereas Lutz reports 10 x 106 cells. We perform RBC lysis on the initial bone marrow cells whereas the protocol by Lutz et al. does not and so this step may be optional. Variations in the percent of CD11c cells as well as numbers and the proportions of macrophages and DCs can arise from different sources or batches of FCS and so it is critical to test new batches of FCS on BMC cultures for consistent experimental results. GM-CSF concentration was varied from 5 ng/ml to 40 ng/ml and this did not significantly affect the cell yield. Cell density may also influence the maturation of the culture. For example, Helft et al. seeded 1 x 107 cells in 4 ml media which produced a greater percent of cells with high MHCII expression19. The time point when cells are harvested can also affect the percentage of populations present in the non-adherent fraction. Cells are typically collected between days 7 – 10 25-27, but some studies collect BMCs at day 619,23,24. If more cells are needed, culture BMCs in larger 150 mm x 15 mm Petri dishes in 40 ml at the same density. In this case a half-volume media change is done on both day 3 and day 6 (i.e., 20 ml of media removed, cells spun down and replaced with fresh 20 ml media supplemented with 20 ng/ml rGM-CSF). These plates typically generate 3 – 6 x 107 cells per dish at day 7. FL-HA is an important reagent, together with MHCII, to distinguish macrophages from immature and mature DCs in the BMC culture. Thus once it is made, titrate the FL-HA for use on cells known to constitutively bind HA such as alveolar macrophages18 or BW5147 T cells and confirm its specificity using either a HA-blocking CD44 antibody (KM-81, commercially available), unlabeled HA or a CD44 deficient cell. CD11c– Gr1+ cells in the culture can function as an internal control for non HA-binding cells, and a fluorescence minus one control (FMO) or CD44 deficient BMC cultures are highly recommended for accurate setting of the FL-HA binding gate.
Although experiments can be performed using a heterogeneous culture, it is advisable to enrich for the DC or macrophage population using either fluorescence activated cell sorting or antibody coated magnetic beads18 based on the expression of CD11c, Gr1, MHCII, and HA binding. Possible experiments include stimulation with Toll-like receptor agonists such as LPS to measure activation and cytokine production, T cell activation assays, or additional characterization by flow cytometry. For example, after 8 or 24 hr of stimulation, flow cytometry can be used to measure intracellular staining for cytokines and/or surface labeling of co-stimulatory molecules such as CD40 and CD86, as described in18. Intracellular cytokine labeling procedures require pretreatment of the cells with Brefeldin A, which prevents secretion and allows the cytokine to build up inside the cell. For T cell activation assays involving the loading of an antigen such as the OVA peptide by an antigen presenting cell, purified macrophages based on MHCII and HA binding will not activate T cells while immature and mature DCs will18. It should be noted that the sorting procedure alone may activate the dendritic cells to some extent, making it important to include negative controls in all experiments.
As with any in vitro method, there are advantages and disadvantages compared to using in vivo or ex-vivo cells. The disadvantage is that in vitro derived cells may not exactly mimic in vivo cells but they have the advantage that they can be generated in sufficient numbers to allow functional and biochemical analysis, something that is often not feasible with ex-vivo cells. This method of macrophage and DC identification will allow future research to obtain more homogenous cell populations from a culture with previously underappreciated heterogeneity. As purified macrophages from this GM-CSF culture closely resemble alveolar macrophages from the lung18, it provides a convenient source of alveolar-like macrophages for in vitro analysis. For example, these HA binding macrophages could be used to screen for drugs that modify alveolar macrophage function and to investigate mechanisms of Mycobacterium tuberculosis infection and resistance. Given the recent discovery that GM-CSF and M-CSF bone marrow-derived macrophage transplantation can alleviate pulmonary proteinosis in mice28,29, this method to identify macrophages from GM-CSF cultures may help increase the efficacy of this proposed therapy.
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 |