Name: in vitro phagocytosis of myelin debris by bone marrow-derived macrophages
Uploaded: 2017-12-30T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages at JoVE.com

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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			<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>
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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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