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 coll...

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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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	<tbody>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">DMEM</td>
			<td style="width:149px;">GE Healthcare Life Sciences</td>
			<td style="width:115px;">SH30243.01</td>
			<td style="width:225px;">High glucose with L-glutamine, sodium pyruvate</td>
		</tr>
		<tr height="62">
			<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>
		</tr>
		<tr height="41">
			<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>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:180px;">NCTC clone 929 [L cell, L-929, derivative of Strain L]&#160;</td>
			<td style="width:149px;">ATCC</td>
			<td style="width:115px;">CCL-1</td>
			<td style="width:225px;">L929 Cell Line of Conditioned Media Preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">24-well Cell Culture Plates</td>
			<td style="width:149px;">VWR</td>
			<td style="width:115px;">10062-896&#160;</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:180px;">Cell Culture Dish</td>
			<td style="width:149px;">Greiner Bio-One</td>
			<td style="width:115px;">639960</td>
			<td style="width:225px;">Polystyrine, 145/20mm</td>
		</tr>
		<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>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">Fluoro-gel with Tris Buffer&#160;</td>
			<td style="width:149px;">Electron Microscopy Sciences</td>
			<td style="width:115px;">17985-11</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:180px;">Oil Red O</td>
			<td style="width:149px;">Sigma Aldrich</td>
			<td style="width:115px;">O0625</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:180px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="41">
			<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>
		</tr>
		<tr height="62">
			<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>
		</tr>
		<tr height="41">
			<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>
		</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 ...

The overall goal of this protocol is to assess bone marrow-derived macrophage phagocytosis of brain-derived myelin debris. This protocol is designed to answer key questions in the field of neurotrauma. The advantage of this method is that allows for efficient and relatively cost-effective analysis of macrophage responses in vitro, specifically the phagocytic capacity of bone marrow-derived macrophages for myelin debris.This method can easily be adapted to investigate the effects of external modulators. Having saturated the mouse with 70%ethanol, make an opening in the abdominal skin. Pull the skin back to reveal the legs for dissection.Take care not to puncture the peritoneal cavity during this process. Once exposed, remove the legs by cutting at the ankle and the hip. Place the legs in ice-cold PBS supplemented with antibiotics.Repeat for the other leg. Wash the tissue in fresh ice-cold PBS supplemented with antibiotics to dislodge any particulates that may have adhered to the tissue during dissection. A gentle scraping motion applied along at the muscle will dislodge a majority of the unwanted tissue.With a scalpel and tweezers, free the tibia from the muscle and connective tissue. Once freed, remove both ends to expose the marrow cavity. Repeat this process for the femur.Affix a 25-gauge needle to a 20-millimeter syringe filled with complete bone marrow-derived macrophage media. Then flush the bone marrow cavity. The bone will appear white when sufficiently flushed.Agitate the collected marrow aspirates with an 18-gauge needle for 30 to 90 seconds in order to obtain a single cell suspension. Next pass the suspension through a sterile 70-micrometer pore size cell strainer. Evenly divide the cell suspensions in 145-centimeter cell culture dishes.Each mouse will provide enough for a total of three dishes. After seven days of culture, these dishes will typically be between 70 to 80%confluent with mature macrophages. To isolate crude myelin debris from brains, a series of sucrose gradient ultracentrifugation steps are carried out.To begin, 10 to 12 brains are dissected from mice eight to 10 weeks of age. The brains are then homogenized in 0.32 molar sucrose solution, and then layered onto a 0.383 molar sucrose solution cushion to create a discontinuous gradient. Following centrifugation, the crude myelin debris is found at the interface between the layers.This is collected and dispensed into a Tris buffer solution and centrifuged again to pellet the myelin debris. The pellet is resuspended again in Tris buffer solution and split between two tubes for additional cenetrifugation. To begin, remove the entire brain and place it in a dish with ice-cold 0.32 molar sucrose solution.Using sterile surgical scissors, cut the collected brains into pieces approximately five millimeters in size. This step aids in the subsequent homogenization process. Collect the tissue into a 50-milliliter tube.Using a rotary homogenizer, homogenize the tissue into a smooth slurry. Make sure that all large visible brain solids have been thoroughly homogenized. Into an ultracentrifuge tube containing 20 milliliters of 0.83 molar sucrose solution, generate and homogenate, taking care to maintain the separation.Carefully balance each tube using the 0.32 molar sucrose solution. Once finished, transfer the tubes to an appropriate centrifuge rotor and spin for 45 minutes at 100, 000 times gravity, four degrees Celsius. Ensure that acceleration and deceleration are set to their minimum values.Once centrifugation is complete, the myelin debris will be visible between the sucrose density interface. Gently collect the myelin debris. Once collected, add Tris buffer to the myelin debris.Homogenize again with a rotary homogenizer. Divide the homogenate between six clean ultracentrifuge tubes and balance the tubes as needed with additional Tris buffer. Centrifuge the collected myelin debris again at 100, 000 times gravity, four degrees Celsius for 45 minutes, with acceleration and deceleration set to their maximum values.After centrifugation, the myelin will have been pelleted. Using the Tris buffer, resuspend the pellet. Combine the resuspensions and ultracentrifuge again.After centrifugation, a tightly-packed pellet is formed. Decant the supernate. Resuspend the pellet in sterile PBS.Equally divide the resuspended myelin debris into preweighed microcentrifuge tubes. After centrifugation for 10 minutes at 22, 000 times gravity, remove the PBS supernate. After weighing the pellet, resuspend to a concentration of 100 milligrams per milliliter with PBS.The resulting crude myelin debris is now suitable for study of phagocytosis. CFSE labeling myelin debris can be used to track early internalization as well as intracellular trafficking. Unlabeled myelin debris is compatible with Oil Red-O lipid staining to assess the storage and metabolism of the internalized lipids.If you are using myelin debris that has been stored at negative 80 degrees Celsius, you'll first want to resuspend with a 29-gauge needle. Resuspend 10 milligrams of pelleted myelin debris in 200 microliters of PBS. Then add two microliters of five-millimolar CFSE solution.Pipette to mix. After a 30 minute incubation, protected from light and subsequent washing, resuspend the myelin debris to 100 milligrams per milliliter with PBS. Following culture and fixation with 4%paraformaldehyde, add 100%propylene glycol to each well.After incubating for five minutes at room temperature, add Oil Red-O staining solution to each well. Incubate for eight minutes at 60 degrees Celsius with gentle agitation. Gently tilt the plate to allow for complete aspiration of the Oil Red-O stain.To each well add 85%propylene glycol. After five minutes of incubation at room temperature, wash wells with PBS and stain cells with your choice of nuclear dye. Representative light microscopy images of bone marrow cells during culture.24 hours after initial seeding, few adherent cells are present. However, in the presence of macrophage colony-stimulating factor, M-CSF, leukocyte precursor cells begin to undergo differentiation. After seven days of culture in the presence of M-CSF, precursor cells are fully differentiated into mature bone marrow-derived macrophages capable of phagocytosis.Using this method, a single eight-to 10-week-old C57 black 6J mouse will typically yield 18 to 24 million bone marrow macrophages. To visualize internalization, CFSE-labeled myelin debris is added to bone marrow-derived macrophage cultures. Cells were treated with one milligram per milliliter CFSE-labeled myelin debris for one hour prior to washing and fixation with 4%paraformaldehyde.Internalized myelin debris can be visualized using standard GFP filter sets on an epifluorescent-capable microscope. To demonstrate how myelin lipid accumulation changes over time, macrophages were treated with unlabeled myelin debris for 90 minutes, then washed and fixed at various time points. Immediately following washing, few lipid droplets are visible.However, as time progresses, more lipid droplets become visible, reaching a maximum approximately 24 hours after washing. Macrophages will begin to metabolize for efflux to lipid stores, reducing the number of stainable droplets. To quantify Oil Red-O staining, five randomly-obtained images from each time point sample were obtained using an epifluorescent-capable microscope.The Oil Red-O-stained area was determined for each well using images captured in the DsRed channel. The total number of cells is determined by counting the number of nuclei present in each field. This graph shows the resulting quantification.A steady increase in Oil Red-O-positive signal is typical, as the bone marrow-derived macrophages convert internalized myelin lipids into neutral lipids. A decrease in Oil Red-O-positive signal follows the peak at 24 hours as macrophages begin to metabolize or efflux the lipids. Having watched this video, you should have a good idea of how to isolate and culture primary bone marrow-derived macrophages, as well as how to study their interaction with freshly isolated brain-derived myelin debris.The most important part of these procedures is the proper maintenance of aseptic technique at all times. Because macrophages can robustly respond to small quantities of pathogen-associated molecules, it is critical to minimize any potential interaction that can bias results. The main advantage of these techniques is that they make us of biologically relevant primary cells and fresh myelin debris, while eliminating the amount of specialized equipment needed.Additionally, with some user optimization, these methods can be easily adapted to address a wide variety of questions regarding the response of bone marrow-derived macrophages to myelin debris. By making use of both florescently-labeled myelin debris and Oil Red-O staining of myelinated macrophage, it is possible to investigate potential therapeutic interventions for patients suffering from a variety of neurotrauma. The overall goal of such work is for remote macrophage-mediated cellular debris clearance to promote inflammation resolution.

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Neuroscience

12.3K visualizações.  Florida State University. The overall goal of this protocol is to assess bone marrow-derived macrophage phagocytosis of brain-derived myelin debris. This protocol is designed to answer key questions in the field of neurotrauma. The advantage of this method is that allows for efficient and relatively cost-effective analysis of macrophage responses in vitro, specifically the phagocytic capacity of bone marrow-derived macrophages for myelin debris.This method can easily be adapted to investigate the effects of ext...