Name: generation and identification of gm-csf derived alveolar-like macrophages and dendritic cells from mouse bone marrow
Uploaded: 2016-06-25T14:00:00.000+00:00
Duration: 11 min 5 s
Description: Watch this Scientific Journal Video about Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow at JoVE.com

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&#39;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.

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
<ol>
	<li>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.</li>
	<li>Prepare dissection tools by soaking in 70% ethanol (1 pair of dissection scissors, 2 pairs of forceps), Hank&#8217;s Balanced Salt Solution with 5% fetal calf serum (HBSS-FCS), sterile Petri dishes (100 mm x 15 mm), 1 ml syringes, 26&#189; gauge needles, conical collection tubes (15 ml or 50 ml), and paper towels in the BSC.</li>
	<li>Euthanize the mouse using protocols approved by the institution&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Attach the 26&#189; 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.</li>
	<li>Pass any visible clumps of bone marrow through the syringe a few times to form a single cell suspension.</li>
	<li>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 &#176;C.</li>
</ol>
2. Plating and Culturing of Bone Marrow Cells (BMCs)
<ol>
	<li>Prepare the following reagents and supplies.
	<ol>
		<li>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.</li>
		<li>Prepare BMC media by adding 10% FCS, 20 mM HEPES, 1x nonessential amino acid, 55 &#181;M 2-mercaptoethanol, 50 U/ml penicillin and streptomycin, 1 mM sodium pyruvate, and 2 mM L-glutamine to RPMI medium 1640.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;l of cell suspension and dilute it with 70 &#181;l of 0.4% trypan blue, mix well and transfer 10 &#181;l 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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#181;l 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 &#176;C and 5% CO2. This is day 0 in culture.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Collecting and Preparing BMDC and BMDMs for Flow Cytometry
<ol>
	<li>Keep all buffers at 4 &#176;C.</li>
	<li>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.</li>
	<li>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 &#176;C for 5 min, pipette up and down to remove the adherent cells and collect in a separate tube.</li>
	<li>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 &#176;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 &#176;C.</li>
	<li>For flow cytometric analysis, transfer 2 x 105 cells for each sample into 5 ml polystyrene round bottom tubes, add 300 &#181;l of FACS buffer at 4 &#176;C (1x phosphate buffered saline, 2 mM EDTA and 2% bovine serum albumin) to each sample.</li>
	<li>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.</li>
	<li>Resuspend cells in 100 &#181;l 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 &#176;C. Then add 300 &#181;l of FACS buffer to each sample, gently vortex, then spin cells down at 314 x g for 5 min and aspirate the supernatant.</li>
	<li>Resuspend cells in 100 &#181;l of CD11c-PeCy7 at 0.2 &#181;g/ml, Gr1-Pacific Blue at 0.25 &#181;g/ml, MHCII-APC at 0.05 &#181;g/ml, and FL-HA at approximately 2&#160;&#181;g/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.</li>
	<li>Incubate for 20 min at 4 &#176;C to allow binding to the cell surface. Add 300 &#181;l of FACS buffer to each sample, gently vortex, then spin cells down at 314 x g for 5 mins at 4 &#176;C and aspirate the supernatant.&#160;NOTE: If biotinylated antibodies are used in 3.8, repeat 3.8 - 9 with fluorescent-streptavidin.</li>
	<li>Wash cells with another 300 &#181;l 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 &#181;l of FACS buffer containing 0.1 - 0.2 &#181;g 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.</li>
</ol>

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The authors have no financial conflicts of interest.

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&#160; 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...

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.

<table><tbody><tr><td>Flow Cytometer</td><td>BD&amp;nbsp;</td><td>LSR-II</td><td></td></tr><tr><td>Automated Inverted Microscope&amp;nbsp;</td><td>Leica&amp;nbsp;</td><td>DMI4000 B</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Thermo Fisher</td><td>ST-40R</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>Nuaire</td><td>NU-425-600</td><td></td></tr><tr><td>Syringe 1 ml</td><td>BD</td><td>309659</td><td></td></tr><tr><td>26 1/2 Gauge Needle</td><td>BD</td><td>305111</td><td></td></tr><tr><td>50 ml Conical Tube&amp;nbsp;</td><td>Corning</td><td>357070</td><td>*Falcon brand</td></tr><tr><td>Eppendorf tubes (1.5 ml)</td><td>Corning</td><td>MCT-150-C</td><td></td></tr><tr><td>5 ml polystyrene round bottomed tubes</td><td>Corning</td><td>352052</td><td></td></tr><tr><td>Dissection Tools</td><td>Fine Science Tools&amp;nbsp;</td><td>*Various&amp;nbsp;</td><td>*Dissection scissors, dumont forcep and standard forcep&amp;nbsp;</td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Reichert</td><td>1490</td><td></td></tr><tr><td>Sterile 100 mm x 15 mm Petri Dish</td><td>Corning</td><td>351029</td><td>*Falcon brand</td></tr><tr><td>2-Mercaptoethanol</td><td>Thermo Fisher</td><td>21985-023</td><td></td></tr><tr><td>Ammonium Chloride</td><td>BDH</td><td>BDH0208-500G</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Fisher Bioreagents</td><td>BP1600-1</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>B7651-5MG</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134-1KG</td><td>Ethylenediaminetetraacetic acid</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher</td><td>16000-044</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermo Fisher</td><td>14175-095&amp;nbsp;</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher</td><td>15630-080</td><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G8540-100G</td><td></td></tr><tr><td>LPS Ultrapure</td><td>Invivogen</td><td>tlrl-3pelps</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution&amp;nbsp;</td><td>Thermo Fisher</td><td>11140-050</td><td></td></tr><tr><td>Penicillin/Streptomycin 100x</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>BDH</td><td>BDH0268-500G</td><td></td></tr><tr><td>Potassium Chloride</td><td>BDH</td><td>BDH9258-500G</td><td></td></tr><tr><td>Recombinant GM-CSF</td><td>Peprotech</td><td>315-03-A</td><td></td></tr><tr><td>Rooster Comb Sodium Hyaluronate&amp;nbsp;</td><td>Sigma</td><td>H5388-1G</td><td>*Used to make fluoresceinated hyaluronan</td></tr><tr><td>RPMI-1640&amp;nbsp;</td><td>Thermo Fisher</td><td>21870-076</td><td>No sodium pyruvate no glutamine. Warm media to 37&amp;nbsp;&amp;#176;C before using.</td></tr><tr><td>Sodium Chloride</td><td>Fisher&amp;nbsp;</td><td>5271-10&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma</td><td>50876-1Kg</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>P5290-100G</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Fisher Bioreagents</td><td>BP152-5</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Anti-Fc Receptor (unlabeled), Tissue Culture Supernatant</td><td>N/A</td><td>N/A</td><td>Clone: 2.4G2</td></tr><tr><td>Anti-CD11c PeCy7</td><td>eBioscience</td><td>25-0114-82</td><td>Clone: N418</td></tr><tr><td>Anti-Gr-1 efluor450</td><td>eBioscience</td><td>48-5931-82</td><td>Clone: RB6-8C5</td></tr><tr><td>Anti-MHCII APC</td><td>eBioscience</td><td>17-5321-82</td><td>Clone: M5/114.15.2</td></tr><tr><td>Biotinylated Anti-MerTK</td><td>Abcam</td><td>BAF591</td><td>Goat polyclonal IgG</td></tr><tr><td>Streptavidin PE</td><td>eBioscience</td><td>12-4317-87</td><td></td></tr><tr><td>Propidium Iodide</td><td>Sigma</td><td>P4170-25MG</td><td></td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole)</td><td>Sigma</td><td>D9542-5MG</td><td></td></tr></tbody></table>

generation and identification of gm-csf derived alveolar-like macrophages and dendritic cells from mouse bone marrow

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.

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...

Watch this Scientific Journal Video about Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow at JoVE.com

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

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. ...

generation-identification-gm-csf-derived-alveolar-like-macrophages

Research

JoVE Journal

Immunology and Infection

18.0K Views.  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. 

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

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Biology

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

Author Spotlight: Developing a Reproducible Method for Isolating Bone Marrow-Derived Macrophages from Neonatal Mice to Advance Early Life Immune Responses

This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...