Immunology and Infection
Published: June 25th, 2016
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....
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
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.......
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.......
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.....
|Automated Inverted Microscope
|Syringe 1 ml
|26 1/2 Gauge Needle
|50 ml Conical Tube
|Eppendorf tubes (1.5 ml)
|5 ml polystyrene round bottomed tubes
|Fine Science Tools
|*Dissection scissors, dumont forcep and standard forcep
|Sterile 100 x 15 mm Petri Dish
|Bovine Serum Albumin
|Fetal Bovine Serum
|Hank's Balanced Salt Solution
|MEM Non-Essential Amino Acids Solution
|Potassium Phosphate Monobasic
|Rooster Comb Sodium Hyaluronate
|*Used to make fluoresceinated hyaluronan
|No sodium pyruvate no glutamine. Warm media to 37oC before using.
|Sodium Phosphate Dibasic
|Anti-Fc Receptor (unlabeled), Tissue Culture Supernatant
|Goat polyclonal IgG
Copyright © 2024 MyJoVE Corporation. All rights reserved