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>

<ol>
	<li>Wynn, T. A., Chawla, A., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+biology+in+development,+homeostasis+and+disease.">Macrophage biology in development, homeostasis and disease.</a> Nature. 496 (7446), 445-455 (2013).</li><li>Unanue, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-presenting+function+of+the+macrophage.">Antigen-presenting function of the macrophage.</a> Annu Rev Immunol. 2, 395-428 (1984).</li><li>Cavaillon, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+and+macrophages.">Cytokines and macrophages.</a> Biomed Pharmacother. 48 (10), 445-453 (1994).</li><li>Ren, Y., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+the+importance+of+being+eaten.">Apoptosis: the importance of being eaten.</a> Cell Death Differ. 5 (7), 563-568 (1998).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+in+spinal+cord+injury:+phenotypic+and+functional+change+from+exposure+to+myelin+debris.">Macrophages in spinal cord injury: phenotypic and functional change from exposure to myelin debris.</a> Glia. 63 (4), 635-651 (2015).</li><li>Goodrum, J. F., Earnhardt, T., Goines, N., Bouldin, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+myelin+lipids+during+degeneration+and+regeneration+of+peripheral+nerve:+an+autoradiographic+study.">Fate of myelin lipids during degeneration and regeneration of peripheral nerve: an autoradiographic study.</a> J Neurosci. 14 (1), 357-367 (1994).</li><li>Greenhalgh, A. D., David, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+Phagocytic+Response+of+Microglia+and+Peripheral+Macrophages+after+Spinal+Cord+Injury+and+Its+Effects+on+Cell+Death.">Differences in the Phagocytic Response of Microglia and Peripheral Macrophages after Spinal Cord Injury and Its Effects on Cell Death.</a> J Neurosci. 34 (18), 6316 (2014).</li><li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+activates+FAK/Akt/NF-kappaB+pathways+and+provokes+CR3-dependent+inflammatory+response+in+murine+system.">Myelin activates FAK/Akt/NF-kappaB pathways and provokes CR3-dependent inflammatory response in murine system.</a> PLoS One. 5 (2), e9380 (2010).</li><li>Gensel, J. C., Zhang, B. <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+its+role+in+repair+and+pathology+after+spinal+cord+injury.">Macrophage activation and its role in repair and pathology after spinal cord injury.</a> Brain Res. , 1-11 (2015).</li><li>Bourre, J. M., Pollet, S., Daudu, O., Le Saux, F., Baumann, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+consists+of+a+continuum+of+particles+of+different+density+with+varying+lipid+composition:+major+differences+are+found+between+normal+mice+and+quaking+mutants.">Myelin consists of a continuum of particles of different density with varying lipid composition: major differences are found between normal mice and quaking mutants.</a> Biochimie. 59 (10), 819-824 (1977).</li><li>Larocca, J. N., Norton, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Cell Biology. , (2001).</li><li>Norton, W. T., Poduslo, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+in+rat+brain:+method+of+myelin+isolation.">Myelination in rat brain: method of myelin isolation.</a> J Neurochem. 21 (4), 749-757 (1973).</li><li>Wang, X. Q., Duan, X. M., Liu, L. H., Fang, Y. Q., Tan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carboxyfluorescein+diacetate+succinimidyl+ester+fluorescent+dye+for+cell+labeling.">Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling.</a> Acta Biochim Biophys Sin (Shanghai). 37 (6), 379-385 (2005).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77 (6), 499-508 (1999).</li><li>Deutsch, M. J., Schriever, S. C., Roscher, A. A., Ensenauer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+image+analysis+approach+for+lipid+droplet+size+quantitation+of+Oil+Red+O-stained+cultured+cells.">Digital image analysis approach for lipid droplet size quantitation of Oil Red O-stained cultured cells.</a> Anal Biochem. 445, 87-89 (2014).</li><li>Kroner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+and+increased+intracellular+iron+alter+macrophage+polarization+to+a+detrimental+M1+phenotype+in+the+injured+spinal+cord.">TNF and increased intracellular iron alter macrophage polarization to a detrimental M1 phenotype in the injured spinal cord.</a> Neuron. 83 (5), 1098-1116 (2014).</li><li>Trouplin, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow-derived+Macrophage+Production.">Marrow-derived Macrophage Production.</a> J Vis Exp. (81), e50966 (2013).</li><li>Chen, S., So, E. C., Strome, S. E., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Detachment+Methods+on+M2+Macrophage+Phenotype+and+Function.">Impact of Detachment Methods on M2 Macrophage Phenotype and Function.</a> J Immunol Methods. 426, 56-61 (2015).</li><li>Guo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescuing+macrophage+normal+function+in+spinal+cord+injury+with+embryonic+stem+cell+conditioned+media.">Rescuing macrophage normal function in spinal cord injury with embryonic stem cell conditioned media.</a> Mol Brain. 9 (1), 48 (2016).</li><li>Bosco, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+synthetic+matrix+metalloproteinase+inhibitor+reduces+human+mesenchymal+stem+cell+adipogenesis.">A new synthetic matrix metalloproteinase inhibitor reduces human mesenchymal stem cell adipogenesis.</a> PLOS ONE. 12 (2), e0172925 (2017).</li><li>Ram&#237;rez-Zacar&#237;as, J. L., Castro-Mu&#241;ozledo, F., Kuri-Harcuch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+adipose+conversion+and+triglycerides+by+staining+intracytoplasmic+lipids+with+oil+red+O.">Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O.</a> Histochem. 97 (6), 493-497 (1992).</li><li>Koopman, R., Schaart, G., Hesselink, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+oil+red+staining+permits+combination+with+immunofluorescence+and+automated+quantification+of+lipids.">Optimisation of oil red staining permits combination with immunofluorescence and automated quantification of lipids.</a> Histochem Cell Biol. 116 (1), 63-68 (2001).</li><li>Fang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-Induced+Macrophage+Inflammatory+Response+Is+Regulated+by+SHIP.">Lipopolysaccharide-Induced Macrophage Inflammatory Response Is Regulated by SHIP.</a> J Immunol. 173 (1), (2004).</li><li>Martinez, F. 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.

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			<td height="62" style="height:63px;width:180px;">Penicillin-Streptomycin Solution</td>
			<td style="width:149px;">Corning</td>
			<td style="width:115px;">30-002-CI</td>
			<td style="width:225px;">100X Solution</td>
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			<td height="41" style="height:42px;width:180px;">New Born Calf Serum (NCS)</td>
			<td style="width:149px;">Rocky Mountain Biologics</td>
			<td style="width:115px;">NBS-BBT-5XM</td>
			<td style="width:225px;">United States Origin</td>
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			<td style="width:149px;">Greiner Bio-One</td>
			<td style="width:115px;">639960</td>
			<td style="width:225px;">Polystyrine, 145/20mm</td>
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		<tr height="41">
			<td height="41" style="height:42px;width:180px;">CFSE Cell Proliferation Kit</td>
			<td style="width:149px;">Thermo Fisher</td>
			<td style="width:115px;">C34570</td>
			<td style="width:225px;">DMSO for Reconsitution Provided</td>
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			<td height="41" style="height:42px;width:180px;">Fluoro-gel with Tris Buffer&#160;</td>
			<td style="width:149px;">Electron Microscopy Sciences</td>
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			<td style="width:149px;">Sigma Aldrich</td>
			<td style="width:115px;">O0625</td>
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			<td height="21" style="height:21px;width:180px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="42" style="height:42px;">Materials</td>
			<td style="width:149px;">Company</td>
			<td style="width:115px;">Catalog Number</td>
			<td style="width:225px;">Comments</td>
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			<td height="41" style="height:42px;width:180px;">Ultracentrifuge Tubes</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:115px;">326823</td>
			<td style="width:225px;">Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm</td>
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			<td height="62" style="height:63px;width:180px;">SW 32 Ti Ultracentrifuge Rotor</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:115px;">369650</td>
			<td style="width:225px;">SW 32 Ti Rotor, Swinging Bucket, Titanium</td>
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			<td height="41" style="height:42px;width:180px;">Hand Held Rotary Homogenizer</td>
			<td style="width:149px;">Fisher Science</td>
			<td style="width:115px;">08-451-71</td>
			<td style="width:225px;"></td>
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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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12.5K 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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy, which are all associated with muscular atrophy. Primary cultures of spinal MNs have been used widely to demonstrate the involvement of specific genes in such diseases and characterize the cellular consequences of their mutations. This protocol models a primary MN culture derived from the seminal work of Henderson and colleagues more than twenty years ago. First, we detail a method of dissecting the anterior horns of the spinal cord from a mouse embryo and isolating the MNs from neighboring cells using a density gradient. Then, we present a new way of efficiently transfecting MNs with expression plasmids using magnetofection. Finally, we illustrate how to fix and immunostain primary MNs. Using neurofilament mutations that cause Charcot-Marie-Tooth disease type 2, this protocol demonstrates a qualitative approach to expressing proteins of interest and studying their involvement in MN growth, maintenance, and survival.

Modeling Charcot-Marie-Tooth Disease In Vitro by Transfecting Mouse Primary Motoneurons

The goal of this technique is to prepare a highly enriched culture of primary motoneurons (MNs) from murine spinal cord. To evaluate the consequences of mutations causing MN diseases, we describe here the isolation of these isolated MNs and their transfection by magnetofection.

Neurodegeneration of spinal motoneurons (MNs) is implicated in a large spectrum of neurological disorders including amyotrophic lateral sclerosis, ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...