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&#160;</td>
			<td>LSR-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Inverted Microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>DMI4000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</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&#160;</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&#160;</td>
			<td>*Various&#160;</td>
			<td>*Dissection scissors, dumont forcep and standard forcep&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer&#160;</td>
			<td>Richert</td>
			<td>1490</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 100 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&#39;s Balanced Salt Solution</td>
			<td>Thermo Fisher</td>
			<td>14175-095&#160;</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&#160;</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&#160;</td>
			<td>Sigma</td>
			<td>H5388-1G</td>
			<td>*Used to make fluoresceinated hyaluronan</td>
		</tr>
		<tr>
			<td>RPMI-1640&#160;</td>
			<td>Thermo Fisher</td>
			<td>21870-076</td>
			<td>No sodium pyruvate no glutamine. Warm media to 37oC before using.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher&#160;</td>
			<td>5271-10&#160;&#160;</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&#39;,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

17.9K 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

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)1-3. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP4,5, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP.
Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF6-8. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition.
OBJECTIVE: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay.
METHODS: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMR&alpha;) with high purity9-13. The recombinant sGMR&alpha; was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions14,15. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMR&alpha; than those produced by other insects or yeasts.
RESULTS: The cell-free assay system using sGMR&alpha; yielded the data with high plasticity and reliability. GM-CSF binding to sGMR&alpha; was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMR&alpha; is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells.
CONCLUSIONS: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.

We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

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 ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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 ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.