The authors would like to thank Glenn Sanger-Hodgson, Media Specialist at the FSU College of Medicine for all his work in video production, editing, and voice-over.
This work was supported by the National Institutes of Health (R01GM100474 and R01GM072611).

The methods described here and in Section 2 have been approved by the Florida State University Institutional Animal Care and Use Committee (IACUC) and follows the guidelines set forth in the Guide for Care and Use of Laboratory Animals, 8th edition. All animals used in this in this protocol are house in a dedicated laboratory animal facility until use. No in vivo experimentation was performed prior to sacrifice. Animal numbers were based on experimental need using average cell and myelin collections as a guide in order to minimize usage.
NOTE: This protocol describes the generation of bone marrow-derived macrophages (BMDMs) (Section 1), the preparation of fluorescently labeled brain-derived myelin debris (Section 2), the general procedure for analyzing myelin debris phagocytosis (Section 3), and the general procedure for analysis of myelin debris lipid accumulation (Section 4). Reagent preparation, cell harvesting and manipulation, myelin collection and labeling, and assay performance should be completed in a laminar airflow biosafety cabinet.
1. Generation of Primary Bone Marrow-Derived Macrophages
<ol>
	<li>Preparation of complete macrophage culture medium (CMCM) 
	NOTE: Macrophage generation from bone marrow requires the use of macrophage colony-stimulating factor (M-CSF). This preparation utilizes L929 murine fibroblast conditioned media as the source of M-CSF.
	<ol>
		<li>Generate L929 murine fibroblast conditioned media by culturing cells in 145 mm dishes with 50 mL of high glucose (4500 mg/L L-glucose) Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 5% new born calf serum (NCS) and Penicillin/Streptomycin for 7 days.</li>
		<li>Collect media from cultures into 50 mL conical centrifuge tubes and centrifuge for 30 minutes at 3500 x g, 4 &#176;C.</li>
		<li>Filter supernatants through a 0.2 &#181;m syringe filter into a new 50 mL conical centrifuge tube. Conditioned media can be stored at -80 &#176;C for up to 6 months.</li>
		<li>To high glucose DMEM, add 5% vol/vol NCS, 15% vol/vol L929 murine fibroblast conditioned DMEM, and 1% Penicillin/Streptomycin. Media can be stored at 4 &#176;C for 2-3 months. Warm to 37 &#176;C before use.</li>
	</ol>
	</li>
	<li>Collection of primary bone marrow cells and macrophage induction 
	NOTE: Using this protocol one mouse will generate approximately 20 million bone marrow-derived macrophages (BMDMs).
	<ol>
		<li>Euthanize the desired number of 8-10 week old mice using standard CO2 asphyxiation guidelines followed by cervical dislocation.</li>
		<li>Sanitize the animal using 70% (vol/vol) ethanol:H2O, saturating the fur.</li>
		<li>Cut a small opening in the abdomen and carefully pull back the skin to reveal the underlying tissue. Take care to not puncture the peritoneal cavity.</li>
		<li>Remove both hind legs starting at the hip and ending at the ankle. Place the collected tissue into a 100 mm Petri dish containing 5-10 mL sterile phosphate buffered saline (PBS) supplemented with 1% Penicillin/Streptomycin. 
		NOTE: When processing several animals make sure to keep dishes on ice to limit tissue degradation.</li>
		<li>Remove muscle and connective tissue from the bone using a scalpel. Only the femur and tibia are used for cell collection, the fibula can be discarded.</li>
		<li>Expose the marrow cavity of the collected bones by trimming a small section from each end with a scalpel.</li>
		<li>Using a 25g needle, flush the marrow cavities with CMCM into a 50 mL conical centrifuge tube. The bones will appear white once they have been sufficiently flushed.</li>
		<li>Once collection is completed, agitate aspirates with an 18g needle for 30-90 s to generated a single cell suspension. 
		NOTE: An optional red blood cell (RBC) lysis step may be performed at this point. It has recently been suggested that intracellular iron content may influence the effects of myelin debris upon macrophage polarization16. For information regarding the inclusion of a RBC lysis step refer to Trouplin et al.17.</li>
		<li>Filter the suspension through a 70 &#181;m sterile cell strainer into a new 50 mL conical centrifuge tube.</li>
		<li>Seed collected cells evenly into 145 mm cell culture dishes containing 15-20 mL CMCM. Use approximately three culture dishes per mouse sacrificed. Incubate cultures at 37 &#176;C, 5% CO2.</li>
		<li>After 72 hours wash plates once with sterile PBS to remove non-adherent cells, then add 15-20mL fresh CMCM. Incubate cultures for an additional 4 days at 37 &#176;C, 5% CO2. After 7 days of total culture, the hematopoietic bone marrow cells initially isolated will be mature BMDMs (Figure 1).</li>
	</ol>
	</li>
</ol>
2. Generation of Fluorescently Labeled Brain-Derived Myelin Debris
NOTE: All reagents can be stored at 4 &#176;C for up to 1 month.
<ol>
	<li>Preparation of myelin debris collection reagents 
	<ol>
		<li>Prepare Tris&#183;Cl buffer solution.
		<ol>
			<li>To 800mL distilled deionized H2O (ddiH2O) add 20 mL of 1 M Tris&#183;Cl, pH 7.45 (20 mM final concentration) and 20 mL of 100 mM Na2EDTA (2 mM final concentration).</li>
			<li>Adjust pH to 7.45. Adjust volume to 1000 mL with ddiH2O. Filter solution through a 0.2 &#181;m filtration unit.</li>
		</ol>
		</li>
		<li>Prepare 1 M sucrose solution.
		<ol>
			<li>To 100 mL of Tris&#183;Cl buffer solution, add 68.46 g of sucrose. Adjust volume to 200 mL with Tris&#183;Cl buffer solution. Filter solution through a 0.2 &#181;m filtration unit.</li>
		</ol>
		</li>
		<li>Prepare 200 mL of 0.32 M sucrose solution by diluting the 1 M solution with the Tris&#183;Cl buffer solution 0.83:0.16 (vol/vol).</li>
		<li>Prepare 150 mL of 0.83 M sucrose solution by diluting the 1 M solution with the Tris&#183;Cl buffer solution 0.83:0.16 (vol/vol).</li>
	</ol>
	</li>
	<li>Collection of brain-derived crude myelin debris 
	NOTE: The collection method described here is for the isolation of crude myelin debris.
	<ol>
		<li>Euthanize 10-12 mice 8-10 weeks of age using standard CO2 asphyxiation guidelines followed by cervical dislocation.</li>
		<li>Dissect the brains and place them in a 100 mm dish containing 10 mL of 0.32 M sucrose solution. Keep the dish on ice.</li>
		<li>Using sterile surgical scissors cut the brains into pieces approximately 5 mm3 in size.</li>
		<li>Transfer the tissue to a 50 mL conical centrifuge tube and add approximately 30 mL of 0.32 M sucrose solution.</li>
		<li>Homogenize with a sterile hand-held rotary homogenizer, until a smooth solution is achieved.</li>
		<li>Dilute the homogenized brains to a final volume of 90 mL with 0.32 M sucrose solution.</li>
		<li>To six 38.5-mL thin-walled polypropylene ultracentrifuge tubes, add 20 mL of 0.83 M sucrose solution.</li>
		<li>Gently add the homogenized brain solution to the top of the 0.83 M sucrose solution, taking care not to mix the two layers.</li>
		<li>Balance each tube with the 0.32 M sucrose solution.</li>
		<li>Centrifuge at 100,000 x g for 45 min at 4&#176;C using an appropriate pre-cooled ultracentrifuge rotor. Set rotor acceleration and deceleration to their minimum values to reduce myelin debris loss.</li>
		<li>Collect the myelin debris from the interface of the two sucrose densities. 
		NOTE: Debris should appear as a white band towards the center of the tube.</li>
		<li>Combine the crude myelin debris into a 50 mL conical centrifuge tube and adjust the volume to approximately 35 mL using Tris&#183;Cl buffer solution.</li>
		<li>Homogenize the crude myelin debris with a sterile hand-held rotary homogenizer for 30-60 s.</li>
		<li>Evenly divide the suspension between 6 clean ultracentrifuge tubes and balance with an appropriate volume of Tris&#183;Cl buffer solution.</li>
		<li>Centrifuge at 100,000 x g for 45 min at 4&#176;C using an appropriate pre-cooled ultracentrifuge rotor. Set rotor acceleration and deceleration to their maximum values.</li>
		<li>As solid white pellets will now be visible, discard the supernatant and re-suspend the pellets in 10-15 mL of Tris&#183;Cl buffer solution.</li>
		<li>Evenly divide the suspension between 2 clean ultracentrifuge tubes and balance with an appropriate volume of Tris&#183;Cl buffer solution.</li>
		<li>Centrifuge again at 100,000 x g for 45 min at 4 &#176;C using an appropriate pre-cooled ultracentrifuge rotor. Set rotor acceleration and deceleration to their maximum values.</li>
		<li>Discard the supernatant and resuspend the pellets in 5-6 mL of sterile PBS and divide the suspension between an appropriate number of pre-weighed 1.5 mL micro-centrifuge tubes.</li>
		<li>Centrifuge at 22,000 x g for 10 min at 4 &#176;C.</li>
		<li>Discard the supernatant and determine the weight of the myelin debris pellets.</li>
		<li>Re-suspend the pellets in PBS to a final concentration of 100 mg/mL. 
		NOTE: Ten to twelve brains is enough to produce 10-15 mL of 100 mg/mL myelin debris. Myelin debris can be store at -80 &#176;C for 6 months.</li>
	</ol>
	</li>
	<li>Myelin debris fluorescent labeling
	<ol>
		<li>Prepare the 50 &#181;M carboxyfluorescein succinimidyl ester (CFSE) solution immediately before use.
		<ol>
			<li>Using sterile PBS, dilute a 5 mM stock solution of CFSE prepared with 100% dimethyl sulfoxide (DMSO) to a final working concentration of 50 &#181;M.</li>
			<li>Filter solution through a 0.2 &#181;m syringe filter.</li>
		</ol>
		</li>
		<li>Thaw the desired amount of 100 mg/mL myelin debris and re-suspend with a sterile 29g needle. 
		NOTE: Freezing the myelin debris will cause it to drop out of solution requiring resuspension before use.</li>
		<li>Transfer myelin debris to a pre-weighed 1.5 mL micro-centrifuge tube.</li>
		<li>Centrifuge at 14,800 x g for 10 min at 4&#176;C. Discard the supernatant.</li>
		<li>Resuspend the myelin debris in 200 &#181;L of CFSE solution per 100 &#181;L myelin debris pelleted.</li>
		<li>Incubate for 30 min at room temperature (RT) protected from light.</li>
		<li>Centrifuge at 14,800 x g for 10 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Resuspend the pellet in 600-800 &#181;L of wash buffer (0.2 &#181;m filter sterilized 100 mM glycine in PBS).</li>
		<li>Centrifuge at 14,800 x g for 10 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Repeat steps 2.3.8-2.3.9 twice more.</li>
		<li>After the final wash, determine the weight of the myelin debris pellet and re-suspend to 100 mg/mL with sterile PBS. 
		Note: Labeled myelin debris can be store at -80 &#176;C for up to 6 months.</li>
	</ol>
	</li>
</ol>
3. Myelin Debris Phagocytosis Assay
NOTE: The following is the basic method for observing phagocytosis of fluorescently labeled myelin debris. Addition of other treatments and experimental conditions will need to be optimized by the investigator.
<ol>
	<li>Preparation of treatment plates
	<ol>
		<li>For each 145 mm plate, collect mature BMDMs into a 15 mL conical centrifuge tube using 5-6 mL of 10 mM ethylenediaminetetraacetic acid (EDTA) in Dulbecco&#39;s phosphate-buffered saline (dPBS) (pH 7.4). 
		NOTE: Do not use trypsin as it may alter the activation state of the cells18.</li>
		<li>Add an equal volume of pre-warmed CMCM to each tube of collected cells.</li>
		<li>Centrifuge at 180 x g for 8 min at 20 &#176;C. Discard the supernatant.</li>
		<li>Re-suspend cells in CMCM and count.</li>
		<li>To each well of a 24-well cell culture plate, add 1x105 cells in 1 mL of CMCM.</li>
		<li>Incubate at 37 &#176;C, 5% CO2 for 24 h.</li>
	</ol>
	</li>
	<li>Myelin debris treatment and analysis
	<ol>
		<li>Add 10 &#181;L of 100 mg/mL CFSE labeled myelin debris to each well (1 mg/mL final concentration).</li>
		<li>Incubate for 1-3 hours at 37 &#176;C, 5% CO2.</li>
		<li>Wash plate 3 times with sterile PBS to remove non-engulfed myelin debris.</li>
		<li>Add 400 &#181;L of 4% paraformaldehyde to each well. Incubate for 30 min at RT.</li>
		<li>Wash plate 2 times with sterile PBS.</li>
		<li>Add 400 &#181;L of Hoechst 33258 solution to each well. Incubate for 5 min at RT protected from light.</li>
		<li>Wash plates 2 times with sterile PBS.</li>
		<li>Add 200 &#181;L of Fluoro-gel with Tris buffer to each well to reduce photobleaching.</li>
		<li>Image cells using an inverted epi-fluorescent capable microscope. 
		NOTE: The excitation and emission wavelengths of CFSE are 494 nm and 521 nm, respectively.</li>
		<li>Divide the number of CFSE positive cells by the number of nuclei to determine relative myelin debris uptake.</li>
		<li>Prior to imaging, maintain plates for up to 7 days at 4 &#176;C protected from light.</li>
	</ol>
	</li>
</ol>
4. Quantification of Intracellular Lipids Via Oil Red-O Staining
NOTE: The following is the basic method for observing intracellular myelin-debris-derived lipids. The use of CFSE labeled myelin debris is not recommended for fluorescent quantification of ORO staining due to spectral overlap. Addition of other treatments and experimental conditions will need to be optimized by the investigator.
<ol>
	<li>Preparation of staining solutions
	<ol>
		<li>Prepare 0.5% Oil Red-O (ORO) staining solution.
		<ol>
			<li>Slowly add 100 mL of 100% propylene glycol (1,2-Propanediol) to 0.5 g of ORO (1-([4-(Xylylazo)xylyl]azo)-2-naphthol) while stirring.</li>
			<li>Heat solution at 95 &#176;C for 15 min or until all large particles are dissolved. 
			NOTE: Do not heat over 100 &#176;C.</li>
			<li>Filter solution through a piece of filter paper while still warm into a new container.</li>
			<li>Allow solution to cool overnight at RT. Solution can be stored for up to 1 year at RT.</li>
		</ol>
		</li>
		<li>Prepare 85% propylene glycol solution.
		<ol>
			<li>Add 85 mL of 100% propylene glycol to 15 mL of ddiH2O. Stir until mixed. Solution can be stored for up to 1 year at RT.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of treatment plates
	<ol>
		<li>Prepare a 24-well plate following the method outlined in Section 3.</li>
		<li>Incubate at 37 &#176;C, 5% CO2 for 24 h.</li>
	</ol>
	</li>
	<li>Myelin debris treatment 
	<ol>
		<li>Add 10 &#181;L of 100 mg/mL myelin debris to each well (1 mg/mL final concentration).</li>
		<li>Incubate for 1-3 h at 37 &#176;C, 5% CO2.</li>
		<li>Wash plate 3 times with sterile PBS to remove non-digested myelin debris. 
		Note: At this point, plates can either undergo immediate fixation or fresh CMCM can be added, and cells returned to incubation for additional time points.</li>
		<li>Add 400 &#181;L of 4% paraformaldehyde to each well. Incubate for 30 min at RT.</li>
		<li>Wash plate 2 times with sterile PBS then procced to staining.</li>
	</ol>
	</li>
	<li>ORO staining and analysis
	<ol>
		<li>Wash fixed plate 3 times with ddiH2O.</li>
		<li>Add 400 &#181;L of 100% propylene glycol to each well and incubate for 5 min at RT. 
		NOTE: This will reduce carryover of ddiH2O.</li>
		<li>Aspirate the propylene glycol and add 400 &#181;L of ORO solution to each well.</li>
		<li>Incubate for 8 min at 60 &#176;C.</li>
		<li>Aspirate the ORO solution. Then, add 400 &#181;L of 85% propylene glycol to each well and incubate for 5 minumintes at RT.</li>
		<li>Wash plate 3 times with ddiH2O.</li>
		<li>Add 400 &#181;L of Hoechst 33258 solution to each well. Incubate for 5 min at RT, protected from light.</li>
		<li>Wash plates 2 times with sterile PBS.</li>
		<li>Add 200 &#181;L of Fluoro-gel with Tris buffer to each well.</li>
		<li>Image cells using an inverted epi-fluorescent capable microscope. ORO can be imaged using a standard Texas Red or DsRed filter set.</li>
		<li>Quantify relative lipid retention by determining the ORO positive area in each image field and dividing it by the number of nuclei.</li>
		<li>Maintain plates for 7 days at 4 &#176;C protected from light.</li>
	</ol>
	</li>
</ol>

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O., Sica, A., Mantovani, A., Locati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+and+polarization.">Macrophage activation and polarization.</a> Front Biosci. 13, (2008).</li><li>Mosser, D. M., Edwards, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+full+spectrum+of+macrophage+activation.">Exploring the full spectrum of macrophage activation.</a> Nat Rev Immunol. 8 (12), 958-969 (2008).</li></ol>

The authors have no disclosures.

The procedures described here utilize both freshly isolated crude CNS myelin debris and primary bone marrow-derived macrophages. To reduce animal expenditures, we recommend that both brains and bone marrow cells be harvested from each mouse at the time of sacrifice. Two researchers working together can prepare both materials simultaneously. Alternatively, brains can be stored at -80 &#176;C in PBS supplemented with antibiotics prior to myelin debris isolation. It has been our experience that brains can be maintained in t...

Bone marrow-derived macrophages (BMDMs) are an important link between the innate and adaptive immune systems. As antigen presenting cells (APCs), they can communicate with lymphocytes via both antigen presentation and cytokine release1,2,3. However, as professional phagocytes, their primary function is to clear pathogens, aged cells, and cellular debris1,4. Following a spinal cord injury (SCI), substantial quantities of myelin debris is generated from dying oligodendrocytes, the cell type responsible for CNS axon myelination5. We and others have shown that clearance of myelin debris is primarily the responsibility of infiltrating BMDMs5,6,7. However, within spinal cord injury sites engulfment of myelin debris has been suggested to shift these normally anti-inflammatory cells towards a pro-inflammatory state5,8,9. As key mediators of neuro-inflammation in the injured spinal cord, BMDMs are important clinical targets.
To help investigate the influence of BMDMs in the injured spinal cord, we have developed an in vitro model to directly study how BMDMs respond to myelin debris. To improve biological relevance, both primary murine BMDMs and freshly isolated myelin debris are used in these investigations. As such, the methods presented here also detail the isolation and culture of primary murine BMDMs, and a modified sucrose gradient technique used to isolate murine CNS derived myelin debris10,11,12. Myelin debris can be readily labeled with a fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE), to track its internalization by BMDMs. CFSE is well suited for this application because it is non-cytotoxic, and its narrow fluorescent spectrum permits multiplexing with other fluorescent probes13,14. Following phagocytosis, myelin debris lipids are transported through the lysosomes and packaged as neutral lipids into intracellular lipid droplets5. To quantify this intracellular lipid accumulation, we present an Oil Red O (ORO) staining method optimized for quantitative image analysis. This simple staining method produces robust reproducible results and quantification15. These methods facilitate the study of myelin debris phagocytosis and lipid retention with limited specialized equipment.

<table><tbody><tr><td>DMEM</td><td>GE Healthcare Life Sciences</td><td>SH30243.01</td><td>High glucose with L-glutamine, sodium pyruvate</td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Corning</td><td>30-002-CI</td><td>100X Solution</td></tr><tr><td>New Born Calf Serum (NCS)</td><td>Rocky Mountain Biologics</td><td>NBS-BBT-5XM</td><td>United States Origin</td></tr><tr><td>NCTC clone 929 [L cell, L-929, derivative of Strain L]&amp;nbsp;</td><td>ATCC</td><td>CCL-1</td><td>L929 Cell Line of Conditioned Media Preparation</td></tr><tr><td>24-well Cell Culture Plates</td><td>VWR</td><td>10062-896&amp;nbsp;</td><td></td></tr><tr><td>Cell Culture Dish</td><td>Greiner Bio-One</td><td>639960</td><td>Polystyrine, 145/20mm</td></tr><tr><td>CFSE Cell Proliferation Kit</td><td>Thermo Fisher</td><td>C34570</td><td>DMSO for Reconsitution Provided</td></tr><tr><td>Fluoro-gel with Tris Buffer&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>17985-11</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma Aldrich</td><td>O0625</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>Ultracentrifuge Tubes</td><td>Beckman Coulter</td><td>326823</td><td>Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm</td></tr><tr><td>SW 32 Ti Ultracentrifuge Rotor</td><td>Beckman Coulter</td><td>369650</td><td>SW 32 Ti Rotor, Swinging Bucket, Titanium</td></tr><tr><td>Hand Held Rotary Homogenizer</td><td>Fisher Science</td><td>08-451-71</td><td></td></tr></tbody></table>

in vitro phagocytosis of myelin debris by bone marrow-derived macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

Treatment of BMDMs with CFSE labeled myelin debris should yield clear internalization (Figure 2). While a 3-hour interaction time is sufficient for BMDMs to phagocytose enough added myelin debris for robust downstream detection, intracellular accumulation can be observed with as little as 1 hour of interaction. However, some myelin debris may still be present on the cell surface after washing. This may be due to insufficient washing, or particles not being fully internalized during the early...

Watch this Scientific Journal Video about In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages at JoVE.com

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

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12.6K Views.  Florida State University. We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

Video: In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Immunology and Infection

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 ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

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

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

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

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

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

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

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

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

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Phagocytic Assay Evaluation of Neonatal Bone Marrow Derived Macrophages 

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

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

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

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

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

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Summary

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Chapters in this video

Depletion and Reconstitution of Macrophages in Mice

Bone Marrow-derived Macrophage Production

Study of Phagolysosome Biogenesis in Live Macrophages

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

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

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

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