L.K. is the recipient of the University of British Columbia 4-year fellowship (4YF) graduate studentship award. L.M.S. is the recipient of a Canadian Association of Gastroenterology/ Crohn's and Colitis Canada/ CIHR New Investigator Salary Award and is a Biomedical Scholar of the Michael Smith Foundation for Health Research. This work was supported by a Project Grant from Canadian Blood Services, in collaboration with the Canadian Institutes of Health Research (grant # CIHR2016-LS).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of British Columbia.
1. Bone Marrow Macrophage Derivation and Activation with Antibodies
<ol>
	<li>Perform euthanasia using CO2 asphyxiation.
	<ol>
		<li>Place mouse in induction chamber. Euthanize mouse with 5% isoflurane anesthesia, for 60 - 90 s, until immobile and breathing is deep and slow.</li>
		<li>Turn off isoflurane anesthesia and administer 6 - 8 L/min of CO2 until mouse has stopped breathing. Leave mouse with CO2 on for at least another 5 min. Verify that there is no longer a heartbeat or respiration. Perform a cervical dislocation to ensure death. 
		NOTE: 8 - 12 weeks old mice provide the highest yield of macrophages.</li>
	</ol>
	</li>
	<li>Spray mouse legs with 70% ethanol, and pin them with arms and legs stretched out in the supine position over a foam board. With scissors and forceps to hold the skin, make a shallow cut (0.2 cm) to remove the skin and fur from the surface of each of the hind legs. 
	NOTE: Perform this procedure and all tissue culture manipulations in a sterile hood.</li>
	<li>Trim muscle to make tibias and femurs visible. Remove the tibias and femurs, by cutting the bone and muscles just below the hip joint and above the ankles. Trim as much muscle off as possible.</li>
	<li>Spray bones with 70% ethanol and wait 1 min for it to evaporate, then place them in 6 well plate of bone flush medium (Iscove&#39;s modified Dulbecco&#39;s medium (IMDM), 10% fetal bovine serum (FBS)).</li>
	<li>Trim the ends of the bones by cutting 0.1 cm of bone off the ankle and top of the femur so that the bone cavity is exposed. Ensure that the marrow is visible and a 26 gauge needle can be placed in the bone cavity.</li>
	<li>Cut the bones and muscles to separate the tibias and femurs from the knee joints. Insert a 26 gauge needle attached to a 10 mL syringe into the cavity. Flush the bone marrow into a 50 mL conical tube with 5 mL of bone flush medium. Flush two tibias and two femurs, from one mouse, into each 50 mL tube.</li>
	<li>Pipette up and down several times or vortex the marrow at a slow speed to gently disperse clumps. Strings of marrow should be broken up into small (less than 0.5 mm) flecks of marrow. Fill the conical tube to 50 mL with bone flush medium. Pipette contents of the conical tube into a 75 cm2 tissue culture treated flask, and incubate at 37 &#176;C, 5% CO2 for 1 h. 
	NOTE: Mature macrophages and mesenchymal cells will become adherent to the flask and be discarded, while the desired hematopoietic progenitors remain in suspension.</li>
	<li>Pipette the medium with hematopoietic progenitors into a 50 mL conical tube. Centrifuge the tube at 300 x g at room temperature for 5 min. Discard the supernatant and resuspend the pellet in 5 mL of macrophage colony stimulating factor (MCSF) culture medium (IMDM, 10% FBS, 5 ng/mL MCSF, 100 U/mL penicillin/streptomycin, and 150 &#956;M monothioglycerol (MTG)).</li>
	<li>Count cells using a hemocytometer18. Add medium to resuspend cells to a concentration of 0.5 x 106 cells/mL, 1.5 x 107 cells/flask in 30 mL of medium, and pipette up and down gently. Pipette 30 mL of the suspension into a new 75 cm2 tissue culture treated flask.</li>
	<li>On day 4 and day 7 after initial culture in step 1.9, verify that the cells are adherent and slightly branched using an inverted phase contrast bright field microscope. Discard the medium containing the non-adherent cells. Wash the cells with IMDM one time and add 30 mL of MCSF culture medium.</li>
	<li>When the cells are mature by day 10 (&#62;95% positive for F4/80 and Mac-1), verify again that the cells are adherent and slightly.</li>
	<li>To plate cells for stimulations, remove the culture medium from the flask and add 8 mL of enzyme free, EDTA-based cell dissociation buffer (Table of Materials). Incubate the cells for 5 min at 37 &#176;C, 5% CO2.</li>
	<li>Scrape cells gently, with a small amount of pressure, using a sterile cell scraper. Check in the microscope that cells have detached from the flask surface. Pipette dissociated cells into a 50 mL conical tube. Rinse the flask 3 times with 5 mL of IMDM and pool the rinse solution with the cells that were harvested. Centrifuge cells at 300 x g at room temperature for 5 min.</li>
	<li>Resuspend the cell pellet in 3 mL of MCSF culture medium per 50 mL conical tube. Count viable cells using a hemocytometer18. Resuspend cells at a concentration of 1 x 106 cells/mL and plate 100 &#956;L of cell suspension (1 x 105 cells/well) per well of a 96-well tissue culture treated, flat bottom plate. 
	NOTE: Bones from one mouse will generate enough cells for 100 wells. If desired, 1 mL of cells can be plated in a 6-well plate (tissue culture treated, flat bottom). A larger number of cells (1 x 106 cells) may be useful for western blot analyses.</li>
	<li>Once adherent, stimulate duplicate or triplicate wells each with 10 ng/mL of LPS in IMDM, 30 mg/mL of IVIg, or IVIg + LPS. Doses of LPS or IVIg can be titrated to optimize responses. Leave duplicate or triplicate wells as unstimulated controls. Incubate them for 24 h (37 &#176;C, 5% CO2). 
	NOTE: Re-plated cells should adhere to tissue culture wells within 1 h.</li>
	<li>Collect cell supernatants from each well into individual 1.7 mL microcentrifuge tubes and remove any particulate matter by spinning at 10,000 x g for 5 min.</li>
	<li>Remove the cell supernatant and avoid disturbing the pellet. Place the clarified cell supernatant in a sterile microcentrifuge tube. 
	NOTE: Cell supernatants can be assayed for cytokines, IL-10, and cytokine subunit, IL-12/23p40, by enzyme-linked immunosorbent assay (ELISA) immediately, or stored at -80 &#176;C long term. Follow ELISA protocol from commercially available kit (Table of Materials). Other pro-inflammatory cytokines can be assayed, such as IL-6 and TNF, as they are also reduced by co-treatment with IVIg + LPS stimulation compared to stimulation with LPS alone. After stimulation, adherent cells can be prepared for techniques such as western blotting or quantitative polymerase chain reaction (Q-PCR) 19,20.</li>
</ol>
2. Challenging Mice In Vivo with IVIg and Harvesting Peritoneal Macrophages
<ol>
	<li>Weigh each mouse. Calculate the volume of IVIg (100 mg/mL stock concentration) needed to provide a final dose of 2.5 g/kg body weight for each mouse. 
	NOTE: For example, for a 20 g mouse, 500 &#181;L of IVIg is required.</li>
	<li>Draw the required amount of IVIg into a sterile 1 mL syringe fitted with a sterile 26 gauge needle. For control mice that will not receive IVIg, administer an equivalent dose volume of sterile phosphate buffered saline (PBS), pH 7.4 instead of IVIg.</li>
	<li>Scruff the mouse with one hand by grabbing its skin at the back of the neck with your thumb and index finger and holding its tail and hind legs with your remaining fingers. Tilt its body towards the ground.</li>
	<li>Place the needle at a 30 - 40&#176; angle relative to the mouse&#39;s abdomen, in the lower right quadrant, bevel up, and insert 1.5 cm of the needle. Inject the IVIg or PBS intraperitoneally. Wait 1 h.</li>
	<li>Euthanize mice with 5% isoflurane anesthesia followed by 6 - 8 L/min CO2 asphyxiation (see steps 1.1.1 - 1.1.2), or according to institution&#39;s ethical guidelines.</li>
	<li>Pin the mouse to a foam board, with limbs spread out. Spray the parts with 70% ethanol. 
	NOTE: Perform the procedure in a sterile hood.</li>
	<li>Make a shallow cut in the skin (0.2 cm) with scissors along the midline of the mouse, to avoid cutting the peritoneal cavity. Pull the abdominal skin off the mouse&#39;s belly using forceps, so that the lining of the intact peritoneal wall is visible.</li>
	<li>Fill a 5 mL syringe with 5 mL of sterile PBS (pH 7.4). Insert a 25 or 27 gauge needle at the top of the cavity from either the left or right side toward the center of the mouse with the bevel of the needle up. 
	NOTE: Place the needle carefully in the mouse so that you do not inject PBS into the organs, but rather, into the peritoneal space.</li>
	<li>Push the fluid into the peritoneal cavity. Pull the needle out of the cavity and massage the peritoneum for 10 s to dislodge any cells. Insert needle at the top of the cavity and avoid organs. Collect and place the lavage fluid recovered into a 15 mL conical tube on ice. 
	NOTE: For every 5 mL of fluid injected, approximately 3.75 mL will be recovered.</li>
	<li>Repeat injection and recovery (steps 2.8 - 2.9) 3 more times (total injection volume of 20 mL),to recover 15 mL of lavage fluid in total. Collect lavage from each mouse into a separate 15 mL conical tube. 
	NOTE: After the final injection, it may be easier to extract the fluid from the far left and right sides of the mouse, where fluid has accumulated. The organs will cause less interference with the needle at this position.</li>
	<li>Spin cells down at 300 x g for 5 min at 4 &#176;C. Resuspend the cells in 500 &#956;L of plating medium (IMDM, 10% FBS, and 100 U/mL penicillin/streptomycin) per mouse flushed. Count viable macrophages using a hemocytometer18. 
	NOTE: The macrophages will be slightly larger than the other peritoneal cells. Typical yield is 5 x 105 - 1 x 106 macrophages/mouse using a wild type C57BL/6 mouse. 
	Optional: If a large number of red blood cells are present in the cell pellet, perform a lysis step to remove them, using the 1x red blood cell lysis buffer according to manufacturer&#39;s instructions.</li>
	<li>Resuspend cells in plating medium at 1 x 106 macrophages/mL and plate 100 &#956;L per well in a 96-well flat bottom tissue culture treated plate. 
	NOTE: It may be necessary to pool cells from more than one mouse for an experiment if using triplicate wells or titrating stimulants. For example, if one mouse had 5 x 105 macrophages, 500 &#956;L of plating medium would be required. There would be enough cells for 5 wells, or 1 experiment in duplicate, with one LPS dose.</li>
	<li>Incubate cells for 1 h at 37 &#176;C, 5% CO2 to allow macrophages to become adherent. The adherent cells are the peritoneal macrophages. Remove cell supernatants and non-adherent cells. Rinse the wells twice with 200 &#956;L IMDM (pre-warmed to 37 &#176;C), wait 10 s and slowly tilt the plate. Replace the plating medium. Incubate the cells at 37 &#176;C, 5% CO2 for 30 min prior to stimulation.</li>
	<li>Stimulate the macrophages with 10 ng/mL of LPS in IMDM, or leave unstimulated, as a control. Incubate for 24 h, collect and clarify cell supernatants (see steps 1.16 - 1.17) for IL-10 cytokine analysis by ELISA. Follow ELISA protocol from commercially available kit (Table of Materials). 
	NOTE: After stimulation, adherent cells can be prepared for techniques such as western blot or PCR.</li>
</ol>
3. Challenging Mice in Vivo with IVIg + LPS and Harvesting Peritoneal Macrophages
<ol>
	<li>Weigh each mouse. Calculate the volume of IVIg (100 mg/mL stock concentration) needed to provide a final dose of 2.5 g/kg body weight. Calculate the amount of LPS (100 &#956;g/mL stock concentration in IMDM) needed to provide a final dose of 0.2 &#956;g/g body weight. 
	NOTE: For example, for a 20 g mouse, 500 &#181;L of IVIg stock solution and 40 &#956;L of a 100 &#956;g/mL solution of LPS is required.</li>
	<li>Draw the required amount of IVIg and LPS into a sterile 1 mL syringe fitted with a sterile 26 gauge needle. For control mice that will not receive IVIg, replace an equivalent volume of IVIg with sterile PBS pH 7.4.</li>
	<li>Scruff the mouse with one hand by grabbing its skin at the back of the neck with your thumb and index finger and holding its tail and hind legs with your remaining fingers. Tilt its body towards the ground.</li>
	<li>Place the needle at a 30 - 40&#176; angle relative to the mouse&#39;s abdomen, in the lower right quadrant, bevel up, and insert 1.5 cm. Inject the IVIg + LPS, or PBS + LPS, intraperitoneally as in step 2.4.</li>
	<li>After 1h, harvest peritoneal macrophages by performing steps 2.5 - 2.10.</li>
	<li>Spin cells down at 300 x g for 5 min at 4 &#176;C. Remove 1 mL of recovered lavage fluid and use for IL-10 and IL-12/23p40 analyses by ELISA according to manufacturer&#39;s instructions or freeze at -80 &#176;C for future analyses. 
	NOTE: To ensure accurate comparison between treatments for lavage fluid cytokine analysis, perform 5 mL flushs of the peritoneal cavity 4 times for a final volume of 15 mL. Not all fluid will be recovered from each flush.</li>
	<li>As in step 2.11, resuspend and count viable macrophages. 
	NOTE: The macrophages will be slightly larger than the other peritoneal cells. Typical yield is 5 x 105&#160;-&#160;1 x 106 macrophages/mouse using a wild type C57BL/6 mouse. 
	Optional: If a large number of red blood cells are present in the cell pellet, perform a lysis step according to manufacturer&#39;s instructions to remove them, as in step 2.11.</li>
	<li>As in step 2.12, re-suspend cells in plating medium. 
	NOTE: It may be necessary to pool cells from more than one mouse for an experiment. For example, if one mouse had 5 x 105 macrophages, 500 &#956;L of plating medium would be required.</li>
	<li>As in 2.13, incubate cells for 1 h at 37 &#176;C 5% CO2 to allow macrophages to become adherent.</li>
	<li>Collect and clarify conditioned medium, as per steps 1.14 - 1.15, from all peritoneal cells. 
	NOTE: Samples may be used immediately or frozen and stored at -80 &#176;C for cytokine analysis by ELISA.</li>
	<li>Perform step 2.11, but incubate cells for 24 h unstimulated. Collect and clarify cell supernatants, as per steps 1.16 - 1.17, for cytokine analysis by ELISA. 
	NOTE: Cells can be prepared for techniques such as western blot or PCR, as in steps 1.16 - 1.17 and 2.14.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Macrophage activation states play an important role in both tissue homeostasis and disease22. Macrophages can have distinct as well as overlapping activation states, depending on cues in their microenvironment3. They have distinct roles in all stages of the inflammatory response: defense against pathogens, wound healing and tissue restitution, and also have a distinct anti-inflammatory activation state that is important for turning off the inflammatory response<sup class="x...

Macrophages are innate immune cells, which play multiple roles in the immune response to infection or injury. Macrophages are responsible for initiating an immune response to infection or tissue damage, stopping the inflammatory response, and promoting the healing response1. Examples of the three best studied macrophage activation states are: 1) macrophages treated with interferon gamma (IFN&#947;) and bacterial lipopolysaccharide (LPS), designated M(IFN&#947; + LPS), which contribute to the inflammatory response; 2) macrophages stimulated with interleukin 4 (IL-4), M(IL-4), which are associated with the healing response; 3) macrophages stimulated with immune complexes (IC) and LPS, M(IC + LPS), which have the ability to turn off the inflammatory response2,3. M(IC + LPS) are distinct from M(IL-4) wound healing macrophages, and do not express the enzyme arginase (Arg-1) or FIZZ14. The best marker for these anti-inflammatory macrophages is their cytokine production5. Macrophages have multiple roles in maintaining health, but also contribute to inflammatory diseases and cancer3. Because of this, macrophages are a key therapeutic target for the treatment of a wide variety of diseases. It is important to investigate the effects of antibodies on their activation state to develop macrophage-based treatments for disease.
The focus of this paper is on the use of murine bone marrow derived macrophages (BMDMs) and peritoneal macrophages to test the effect of antibody drugs on inflammatory responses in vitro and in vivo. Recently, there have been multiple studies showing the effects of antibodies on macrophage activation6,7,8. Macrophages co-activated with immune complexes, which are antibodies complexed with an antigen, and LPS, a normally inflammatory stimulus, produce very high levels of the anti-inflammatory cytokine, IL-10, and very low levels of the pro-inflammatory cytokine, interleukin 12 (IL-12)9. In addition, infliximab, a monoclonal antibody against TNF&#945;, has been found to work, in part, by inducing anti-inflammatory macrophages through its fragment crystallizable (Fc) region7. We have reported that IVIg + LPS induce anti-inflammatory macrophage activation that is similar to M(IC + LPS), wherein co-stimulated macrophages produce large amounts of IL-10 and low amounts of the pro-inflammatory cytokine subunit Interleukin 12 or 23 p40 (IL-12/23p40), interleukin- 6 (IL-6), and TNF8. IVIg is a drug comprised of polyclonal antibodies, primarily IgG, which has been pooled from the blood of more than 1,000 donors10. It is used to treat a wide variety of immunological diseases, such as idiopathic thrombocytopenic purpura and chronic demyelinating polyneuropathy, but its mechanism of action is not completely understood11. The effects of antibody based drugs on macrophage activation can be assessed using methods described herein.
The effects of specific biologics on macrophage activation can be tested in BMDMs and peritoneal macrophages. Using these macrophage sources permits assessment of primary cells. Preliminary testing of antibodies on cultured primary cells requires less time and monetary investment than other time consuming and expensive disease models. By injecting a drug into a healthy mouse in vivo, and isolating the cells and analyzing them ex vivo, one can determine if studies are warranted to assess whether treatment with biologics affects macrophage activation in disease models.
With few studies testing the effect of biologic therapies on macrophage activation in vitro and in vivo directly, our techniques provide an advantage over alternative techniques. Current techniques involve testing biologic drug effects on mixed cell populations in vitro, such as the effect of infliximab in a mixed lymphocyte reaction (MLR) or IVIg on human macrophages in peripheral blood mononuclear cells, where the effect cannot be attributed to a specific cell type7,12. Using BMDMs and peritoneal macrophages is advantageous over using cell lines, such as RAW264.7 cells, which do not produce the pro-inflammatory cytokine, IL-12, in response to LPS8,13. Testing the effect of an antibody-based drug on macrophage responses ex vivo has advantages because cytokine responses can be attributed directly to macrophages, rather than inferring macrophage responses by measuring serum cytokine levels14. BMDMs and peritoneal macrophages can be derived and isolated from genetically modified mice to determine the specific role of a protein in anti-inflammatory macrophage activation. For example, we have used Il10 deficient(-/-) BMDMs to demonstrate that IVIg-induced reduction of pro-inflammatory cytokine production is partially dependent on IL-108. A drug&#39;s mechanism of action can be investigated using western blotting, where the role of specific proteins and signaling events can be determined. Quantitative polymerase chain reaction (qPCR) can be performed on BMDMs or peritoneal macrophages to show patterns of gene expression that result from antibody activation. Disease models in mice can provide information on the potential efficacy of antibody-based biological therapies in models for diseases like inflammatory bowel disease, rheumatoid arthritis, and cancer15,16,17. However, the techniques described here will provide information on the mechanism of action of these biologics by determining whether they induce anti-inflammatory macrophage activity.

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			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">12440053</td>
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			<td height="21" style="height:21px;width:453px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:209px;">Life technologies</td>
			<td style="width:408px;">12483-020</td>
			<td></td>
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			<td height="21" style="height:21px;width:453px;">Recombinant murine macrophage colony stimulating factor (MCSF)</td>
			<td style="width:209px;">Stemcell technologies</td>
			<td>78059</td>
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			<td>88640</td>
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			<td>Enzyme-Free, Hanks&#39;s-based, EDTA</td>
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			<td height="21" style="height:21px;width:453px;">Lipopolysaccharide (LPS)</td>
			<td style="width:209px;">Sigma aldrich</td>
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			<td>From E. coli 0127:B8</td>
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			<td height="21" style="height:21px;width:453px;">IVIg (Gammunex)</td>
			<td style="width:209px;">Grifols</td>
			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
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			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
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			<td>Received from BC Children&#39;s Hospital, Transfusion Medicine</td>
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			<td height="21" style="height:21px;width:453px;">mouse IL-12/23p40 ELISA</td>
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			<td style="width:408px;">555165</td>
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			<td height="21" style="height:21px;width:453px;">anti-Erk1/2 antibody</td>
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			<td style="width:408px;">9101</td>
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			<td height="21" style="height:21px;width:453px;">anti-pp38 antibody</td>
			<td style="width:209px;">Cell signalling technology</td>
			<td style="width:408px;">9211</td>
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			<td height="21" style="height:21px;width:453px;">anti-GAPDH antibody</td>
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			<td style="width:408px;">10R-G109A</td>
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			<td height="21" style="height:21px;width:453px;">26 g needle</td>
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			<td style="width:408px;">305110</td>
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			<td height="21" style="height:21px;width:453px;">1 mL syringe</td>
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			<td style="width:408px;">309659</td>
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			<td style="width:408px;">352070</td>
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			<td height="21" style="height:21px;width:453px;">microcentrifuge tube (1.7 mL)</td>
			<td style="width:209px;">Diamed</td>
			<td style="width:408px;">SPE155-N</td>
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			<td height="24" style="height:24px;width:453px;">75 cm2 tissue culture treated flask</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">353136</td>
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			<td height="21" style="height:21px;width:453px;">Cell scraper</td>
			<td style="width:209px;">BD biosciences</td>
			<td style="width:408px;">353085</td>
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			<td height="21" style="height:21px;width:453px;">Forcepts</td>
			<td style="width:209px;">VWR</td>
			<td>82027-386</td>
			<td>fine tip, dissecting&#160;</td>
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			<td height="25" style="height:25px;width:453px;">Scissors</td>
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			<td>82027-582</td>
			<td>Delicate, 4 1/2&#34;</td>
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			<td height="21" style="height:21px;width:453px;">Brightfield microscope</td>
			<td style="width:209px;">Motic</td>
			<td style="width:408px;">AE31</td>
			<td>Inverted phase contrast</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Scale&#160;</td>
			<td style="width:209px;">Mettler&#160;</td>
			<td style="width:408px;">PE 3000</td>
			<td></td>
		</tr>
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assessment of antibody-based drugs effects on murine bone marrow and peritoneal macrophage activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Murine bone marrow derived macrophages can be cultured from hematopoietic cell precursors in bone marrow aspirates. Bone marrow aspirates pooled from femurs and tibias of one C57BL/6 mouse typically yield 107 bone marrow derived macrophages, making them a convenient source of macrophages for experiments. BMDMs can be used to test antibody based drug responses when challenged with an inflammatory stimulus in vitro. Figure 1 shows that IVIg ...

Watch this Scientific Journal Video about Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation at JoVE.com

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

assessment-antibody-based-drugs-effects-on-murine-bone-marrow

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JoVE Journal

Immunology and Infection

8.1K Views.  University of British Columbia. Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Video: Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

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 ...

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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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 ...