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Summary

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.

Abstract

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.

Introduction

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-CSF^1,2. For BMDCs, the addition of FLT3 ligand to the bone marrow culture gives rise to non-adherent classical and plasmacytoid DCs (CD11c^high/MHCII^high and CD11c^lo, B220⁺ respectively) after 9 days in culture^3,4. In contrast, non-adherent cells generated after 7 to 10 days in culture with GM-CSF alone^5,6, GM-CSF and IL-4⁷, or GM-CSF and FLT3 ligand^8,9 generate BMDCs more closely resembling inflammatory DCs (CD11c^high, MHCII^high 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 DCs^6,11-13, with the presumption that they are homogeneous and any observed variability is due to different stages of development^14,15. Furthermore, studies have found GM-CSF to be an essential growth factor for alveolar macrophage development in vivo^16,17, and can be used in vitro to generate alveolar-like macrophages^16,17,18.

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⁺, MHCII^mid, 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⁺, MHCII^mid/low, FL-HA^high) that was distinct from immature (CD11c⁺, MHCII^mid, FL-HA^low) and mature DCs (CD11c⁺, MHCII^high), 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.

Protocol

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 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 entirety of the protocol. Do not cut the bones under non-sterile conditions. Prepare dissection tools by soaking in 70% ethanol (1 pair of dissection scissors, 2 pairs of forceps), Hank’s Balanced Salt Solution with 5% fetal calf serum (HBSS-FCS), sterile Petri dishes (100 mm x 15 mm), 1 ml syringes, 26½ gauge needles, conical collection tubes (15 ml or 50 ml), and paper towels in the BSC. Euthanize the mouse using protocols approved by the institution's animal research ethics committee. Bring into the BSC, place on top of one or two paper towels and spray the mouse with 70% ethanol. In the BSC, open a sterile Petri dish aseptically, aliquot 10 ml of HBSS-FCS to the base and 1 – 2 ml to the lid. Place the mouse on its stomach with the back facing up and lift the skin around the thoracic sections with fingers of the non-dominant hand. Use the scissors to make an incision where the skin is lifted. Hold on to both ends of the cut, one end with each hand and carefully pull apart the skin around the mouse from the back to the stomach, continue pulling until two ends of the incision meet at the stomach. Flip the mouse to stomach facing up, and with one hand pull the lower half of the skin down towards the leg, keep pulling back the skin until the legs are exposed. Hold the foot of the mouse up and cut the Achilles tendons above the ankle joint and the ligaments connecting the muscles to foot at the lower end of the tibia with scissors. This loosens the foot from the leg bones. While holding the leg up by the foot, cut the muscles and ligaments with scissors all around the knee joint at the lower end of the femur to sever the thigh muscles from the lower leg. Use forceps to gently pull loosened muscles away from the fibular and femoral surfaces. Take care not to sever the femoral artery to avoid heavy bleeding. With one hand holding on to the foot, press down open scissors at the top end of the femur at the hip joint, rotate the leg and pull gently to separate the femur from the hip joint while using the scissors to make the final cut to free the leg from the body. From this point onwards, only hold the tissue with sterile forceps. Hold on to the leg at one end and pull off the foot on the other. NOTE: This should not require much force, as connecting ligaments are cut in step 1.9. Alternatively, cut the tibia just above the ankle where the bone is fused. Hold the leg by the distal end of the femur with one set of forceps and proximal end of the tibia and fibula with another, carefully bend the tibia and fibula in the opposite direction of the knee joint to separate them from the femur and patella. Use forceps to pull down the remaining ligaments and muscles still attached to the lower leg bones. Here, remove the fibula along with the connective tissues with forceps as it is too small to be flushed for bone marrow cells. Place the cleaned tibia on the inverted Petri dish lid. Hold the lower end of the femur with one set of forceps and the patella with another, bend the patella and surrounding tissues in the opposite direction of the joint for removal. Then clean off remaining connective tissues from the femur; pulling them away with forceps, and place it on the inverted Petri dish lid. Discard the patella. Repeat 1.7 – 1.13 with the other leg if needed. Transfer the bones to 1.6 ml microcentrifuge tubes containing 1 ml sterile HBSS-FCS for transportation if required. Place bones on the inverted Petri dish lid, with 1 – 2 ml of HBSS-FCS, use forceps to clean any residual tissue still attached to the tibia and femur, then transfer the bones to the base of Petri dish containing 10 ml of HBSS-FCS. Alternatively, clean the bones and remove attached muscle tissue using a 70% ethanol soaked tissue. Cut each of the bones in half with scissors. Partially shield the Petri dish with a sterile lid while cutting to ensure the bones stay in the dish. Alternatively, cut the end of each bone to allow flushing in the next step. Attach the 26½ G needle on to the 1 ml syringe, and draw up 1 ml HBSS-FCS from the Petri dish. Insert the tip of the needle into the end of a bone piece and flush out the bone marrow cells (soft red tissue inside the bones). Insert the needle into the head of the bone and the open end, until all the red colored marrow is removed. Observe the bones as white in color. Pass any visible clumps of bone marrow through the syringe a few times to form a single cell suspension. Discard the flushed bones. Use a 10 ml pipette to mix the cell suspension well and transfer to the collection tube. Rinse the Petri dish once with 5 ml of HBSS-FCS to collect residual cells in the dish. Place cells on ice. NOTE: The bone marrow cell suspension is still viable if stored for 2 – 3 hr at 4 °C. 2. Plating and Culturing of Bone Marrow Cells (BMCs) Prepare the following reagents and supplies. Prepare red blood cell (RBC) lysis buffer by making 0.84% ammonium chloride in a final solution of 2 mM Tris pH 7.2, then sterilize by filtering through a 0.2 micron filter. Prepare BMC media by adding 10% FCS, 20 mM HEPES, 1x nonessential amino acid, 55 µM 2-mercaptoethanol, 50 U/ml penicillin and streptomycin, 1 mM sodium pyruvate, and 2 mM L-glutamine to RPMI medium 1640. Obtain commercially available recombinant GM-CSF or use GM-CSF containing supernatant from AG8.653 myeloma cells transfected with the GM-CSF gene20. Determine the concentration of GM-CSF in the supernatant by ELISA and add the equivalent of 20 ng/ml to the BMC media. Spin down the bone marrow cells at 314 x g for 5 min. The pellet is red, due to RBCs. In the BSC, aspirate the supernatant using a sterile glass Pasteur pipette attached to vacuum suction, loosen the pellet by tapping, then add 10 ml of RBC lysis buffer. Vortex to resuspend the cells and incubate in RBC lysis buffer at room temperature for 5 min. Add 10 ml of HBSS-FCS then spin cells at 314 x g for 5 min and aspirate the supernatant. Observe the cell pellet as white color. Resuspend cells in desired volume of BMC media. Count the resuspended-cells using a hemocytometer after staining dead cells with 0.4% trypan blue. NOTE: If bone marrow from one leg (single tibia and femur) is used, resuspend in 5 ml of BMC media, take 10 µl of cell suspension and dilute it with 70 µl of 0.4% trypan blue, mix well and transfer 10 µl of the 1/8 cell dilution to the hemocytometer chamber. Count the viable cells in four quadrants: the average of the four quadrant counts x dilution factor x 104 = number of cells per ml. A femur and a tibia from one leg will generally yield approximately 2 – 4 x 107 cells after RBC lysis. Estimate the number of 10 ml dishes of bone marrow cultures needed for downstream experiments based on the number of total cells needed for experiments at the end of the culture. NOTE: Each dish requires 2 x 106 bone marrow cells at initial seeding and yields 5 – 10 x 106 cells at day 7. Aliquot the total number of cells for plating into a new tube, spin down at 314 x g for 5 min, aspirate the supernatant, and resuspend cells at 4 x 106 cells/ml in BMC media. Prepare a new sterile 100 mm x 15 mm Petri dish with 9.5 ml of BMC media containing 200 ng of GM-CSF (recombinant or from GM-CSF containing supernatant). Add 500 µl of the 4 x 106 cells/ml bone marrow cells to the center of the Petri dish for the final concentration of 2 x 105 cells/ml in 10 ml. NOTE: It is important to use a sterile Petri dish not a tissue culture dish. Also, when adding the cells, ensure cells remain concentrated at the center and minimize disturbance to the dishes after seeding and during media changes. Incubate cells at 37 °C and 5% CO2. This is day 0 in culture. On day 3, add 10 ml of fresh BMC media containing 200 ng of GM-CSF to each dish of cells. Take care to minimize disturbance of the cells concentrated in suspension. Total volume of cell culture is now 20 ml. Observe cells under the light microscope at 100X magnification. Observe numerous non-adherent cells (round) and a few adherent cells (flat). Cells in groups of three or four resembling petals of a flower can be seen, indicating proliferation. On day 6 perform a half media change. Carefully remove 10 ml of media into a sterile 50 ml conical tube, spin down at 314 x g for 5 min, aspirate and discard the supernatant. Resuspend the cell pellet in 10 ml of fresh BMC media containing 20 ng/ml of GM-CSF, gently vortex and add to the original cell culture dish for a total volume of 20 ml. Observe cells under the microscope. NOTE: Day 7 culture should be at 70% or greater in confluency with adherent flat cells and dense clouds of round non-adherent cells. 3. Collecting and Preparing BMDC and BMDMs for Flow Cytometry Keep all buffers at 4 °C. Harvest the cells from day 7 to day 10. To harvest the cells, first transfer 10 ml of the culture supernatant to a 50 ml conical collection tube, then tilt the dish vertically by approximately 30 degrees and wash the dish by pipetting the remaining 10 ml of culture from the top of the dish. Repeat this wash 5 times, each time rotating the dish by one third. Transfer the remaining 10 ml of cells to the same collection tube and discard the plate and adherent cells. NOTE: The washes will remove non-adherent and loosely adherent cells; the flat adherent cells are resistant to the washes. Typically, between 5 – 10 x 106 non-adherent cells are expected from one plate at day 7 and 2 – 5 x 106 cells at day 10, although Lutz et al. reported 10 x 106 cells at day 10. The adherent cells are not normally taken. However, if these cells need to be compared to the non-adherent cells, they can be removed as follows. Add 5 ml of 0.7 mM EDTA in 1x PBS, pH 7.4 to the dish after the non-adherent cells are removed, incubate at 37 °C for 5 min, pipette up and down to remove the adherent cells and collect in a separate tube. Spin down the collected cells at 314 x g for 5 min and aspirate the supernatant. Resuspend in 5 ml sterile BMC media at 4 °C, vortex gently to mix, and count using trypan blue and hemocytometer. NOTE: Each dish will yield 5 – 10 x 106 non-adherent cells from a day 7 culture. From now on, perform all steps at 4 °C. For flow cytometric analysis, transfer 2 x 105 cells for each sample into 5 ml polystyrene round bottom tubes, add 300 µl of FACS buffer at 4 °C (1x phosphate buffered saline, 2 mM EDTA and 2% bovine serum albumin) to each sample. Spin cells down at 314 x g for 5 min and aspirate the supernatant. Observe a visible white, opaque pellet at the bottom of the tube. Resuspend cells in 100 µl of unlabeled Fc receptor Ab to block Fc receptor binding (Use 1/10 supernatant from 2.4G2 producing myeloma cell line) and incubate for 20 min at 4 °C. Then add 300 µl of FACS buffer to each sample, gently vortex, then spin cells down at 314 x g for 5 min and aspirate the supernatant. Resuspend cells in 100 µl of CD11c-PeCy7 at 0.2 µg/ml, Gr1-Pacific Blue at 0.25 µg/ml, MHCII-APC at 0.05 µg/ml, and FL-HA at approximately 2 µg/ml to identify macrophages and DCs. NOTE: FL-HA was prepared in house using fluorescein and rooster comb hyaluronic acid sodium salt as previously described21. Incubate for 20 min at 4 °C to allow binding to the cell surface. Add 300 µl of FACS buffer to each sample, gently vortex, then spin cells down at 314 x g for 5 mins at 4 °C and aspirate the supernatant. NOTE: If biotinylated antibodies are used in 3.8, repeat 3.8 – 9 with fluorescent-streptavidin. Wash cells with another 300 µl of FACS buffer, gently vortex to mix and spin down the cells at 314 x g for 5 min. Aspirate the supernatant and resuspend the cells in 200 µl of FACS buffer containing 0.1 – 0.2 µg per ml of propidium iodide or DAPI to label nonviable cells. Cells are now ready for flow cytometric analysis22. NOTE: See Results and Figure 3 for details of the populations and how the cells were gated.

Representative Results

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…

Discussion

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…

Materials

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