Grant sponsor: National Institutes of Health; Grant number: R37 NS041435.

Procedures involving animal subjects have been approved by Institutional Animal Care and Use Committee (IACUC) and Johns Hopkins School of Medicine.
1. Preparation of Assay Plates, Stock Solutions, and OPC Base Media
Note: The rat OPC base (Sato) media described here has been derived from previously published studies12,13. Alternative media formulations may also be compatible with this procedure.
<ol>
	<li>Resuspend poly-L-lysine (PLL) to a concentration of 10 &#181;g/ml in sterile deionized wa...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferating+bipotential+glial+progenitor+cells+in+adult+rat+optic+nerve.">Proliferating bipotential glial progenitor cells in adult rat optic nerve.</a> Nature. 319 (6053), 499-502 (1986).</li><li>Popko, 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=Myelin+deficient+mice:+expression+of+myelin+basic+protein+and+generation+of+mice+with+varying+levels+of+myelin.">Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin.</a> Cell. 48 (4), 713-721 (1987).</li><li>Bourre, J. M., 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=Density+profile+and+basic+protein+measurements+in+the+myelin+range+of+particulate+material+from+normal+developing+mouse+brain+and+from+neurological+mutants+(Jimpy;+quaking;+Trembler;+shiverer+and+its+mld+allele)+obtained+by+zonal+centrifugation.">Density profile and basic protein measurements in the myelin range of particulate material from normal developing mouse brain and from neurological mutants (Jimpy; quaking; Trembler; shiverer and its mld allele) obtained by zonal centrifugation.</a> J Neurochem. 35 (2), 458-464 (1980).</li><li>Baxi, E. G., 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+selective+thyroid+hormone+&#946;+receptor+agonist+enhances+human+and+rodent+oligodendrocyte+differentiation.">A selective thyroid hormone &#946; receptor agonist enhances human and rodent oligodendrocyte differentiation.</a> Glia. 62 (9), 1513-1529 (2014).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60 (4), 555-569 (2008).</li><li>Chen, Y., 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=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat Protoc. 2 (5), 1044-1051 (2007).</li><li>Warringa, R. 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=Hydrocortisone+stimulates+the+development+of+oligodendrocytes+in+primary+glial+cultures+and+affects+glucose+metabolism+and+lipid+synthesis+in+these+cultures.">Hydrocortisone stimulates the development of oligodendrocytes in primary glial cultures and affects glucose metabolism and lipid synthesis in these cultures.</a> Brain Res. 431 (1), 79-86 (1987).</li><li>Tosic, M., Torch, S., Comte, V., Dolivo, M., Honegger, P., Matthieu, 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=Triiodothyronine+has+diverse+and+multiple+stimulating+effects+on+expression+of+the+major+myelin+protein+genes.">Triiodothyronine has diverse and multiple stimulating effects on expression of the major myelin protein genes.</a> J Neurochem. 59 (5), 1770-1777 (1992).</li><li>Xiao, 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=Quetiapine+facilitates+oligodendrocyte+development+and+prevents+mice+from+myelin+breakdown+and+behavioral+changes.">Quetiapine facilitates oligodendrocyte development and prevents mice from myelin breakdown and behavioral changes.</a> Mol Psychiatry. 13 (7), 697-708 (2008).</li><li>Mei, F., 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=Micropillar+arrays+as+a+high-throughput+screening+platform+for+therapeutics+in+multiple+sclerosis.">Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis.</a> Nat Med. 20 (8), 954-960 (2014).</li><li>Deshmukh, V. 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=A+regenerative+approach+to+the+treatment+of+multiple+sclerosis.">A regenerative approach to the treatment of multiple sclerosis.</a> Nature. 502 (7471), 327-332 (2013).</li><li>Najm, F. J., 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=Drug-based+modulation+of+endogenous+stem+cells+promotes+functional+remyelination+in+vivo.">Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.</a> Nature. 522 (7555), 216-220 (2015).</li><li>Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+glial+cells+during+CNS+development.">Defining glial cells during CNS development.</a> Nat Rev Neurosci. 2 (11), 840-843 (2001).</li><li>O'Meara, R. W., Ryan, S. D., Colognato, H., Kothary, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+enriched+oligodendrocyte+cultures+and+oligodendrocyte/neuron+myelinating+co-cultures+from+post-natal+murine+tissues.">Derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues.</a> J Vis Exp. (54), (2011).</li><li>Dugas, J. C., Emery, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+oligodendrocyte+precursor+cells+from+rat+cortices+by+immunopanning.">Purification of oligodendrocyte precursor cells from rat cortices by immunopanning.</a> Cold Spring Harb Protoc. 2013 (8), 745-758 (2013).</li><li>Fletcher, J. M., Lalor, S. J., Sweeney, C. M., Tubridy, N., Mills, K. H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+in+multiple+sclerosis+and+experimental+autoimmune+encephalomyelitis.">T cells in multiple sclerosis and experimental autoimmune encephalomyelitis.</a> Clin Exp Immunol. 162 (1), 1-11 (2010).</li></ol>

The authors have nothing to disclose.

The protocol presented here describes a fast and reliable method for isolating rat OPCs and quantifying in vitro oligodendrogenesis in response to experimental factors. Using positive selection, high yields of viable and pure OPCs are used directly for experimental purposes. This method streamlines the isolation, culturing, and differentiation steps into a 1 week time frame, and fixed cells can be conveniently stored at 4 &#176;C for several weeks without any loss in assay sensitivity.
<p class="jove_content...

Efficient nerve conduction in the mammalian central nervous system (CNS) requires myelination of axons by oligodendrocytes. During development, oligodendrocytes arise from a pool of oligodendrocyte progenitor cells (OPCs) that are thought to migrate from the ventricular zones of the developing forebrain and neural tube1. After migration OPCs differentiate into mature, myelinating oligodendrocytes that not only facilitate efficient impulse propagation, but also provide axons with trophic support2. The adult CNS maintains an abundant population of OPCs that are dispersed throughout the gray and white matter comprising approximately 5-8% of all cells3. Following a demyelinating insult, OPCs migrate to the site of injury, proliferate, and differentiate to replace lost or damaged myelin sheaths on exposed axons. However, in some disease/injury settings, this process is found to be inefficient or can fail completely4. While chronic demyelination is thought to add to the burden of disease, efficient oligodendrogenesis and remyelination may alleviate symptoms5. It has therefore been of interest to study OPCs in vitro to determine the effect of experimental factors on oligodendrogenesis.
Insight into the different phases of oligodendrocyte differentiation has been made possible by the identification of stage specific cell markers. Self-renewing early progenitor cells are defined by their expression of platelet derived growth factor receptor alpha (PDGFR&#945;), neural/glia antigen 2 (NG2) and A2B56-8. As OPCs initiate their differentiation program and withdraw from the cell cycle, they downregulate expression of these markers and begin to express proteins indicative of premyelinating oligodendrocytes including Cyclic-nucleotide 3&#39;-phosphodiesterase (CNPase) and O4. Finally, their differentiation into more mature oligodendrocytes is characterized by the expression of myelin-associated proteins, including myelin-associated glycoprotein (MAG), proteolipid protein (PLP), and myelin basic protein (MBP). MBP is an intracellular peripheral membrane protein and a major component of the myelin sheath. Mice lacking MBP develop a severe phenotype in which CNS myelination is significantly decreased leading to tremors, convulsions and early death9,10. This important role of MBP in myelination has led to its use as a marker of oligodendrocyte differentiation both in vitro and in vivo11.
Quantification of MBP can be achieved using several different methodologies. RT-PCR and Western blot analysis allow for quantification of MBP levels under different treatment regimens. Immunocytochemistry is a common qualitative approach that can also be quantitative when camera-based microscopy approaches are used. Although these systems are reliable and fundamental to the study of oligodendrocyte differentiation, they each have their own disadvantages that limit their use in drug screening. First, the amount of primary OPCs that are needed for RT-PCR and Western blot analysis reduces the number of variables that can be examined simultaneously. While the cell requirement for immunocytochemistry is much lower, substantial time must be devoted to each experiment if quantification is the goal. Numerous images must be captured and then quantified to facilitate experimental analysis. These caveats become important obstacles to consider for studies that require high throughput assessment. Here we describe a method that utilizes fundamental aspects from both the immunocytochemistry and Western blot methodologies for myelin quantification, while significantly reducing both the number of cells required and time to complete quantitative analysis.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:691px;">Dissection Materials</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">PBS without Ca2+ and Mg2+</td>
			<td>Mediatech</td>
			<td>21-040-CV</td>
			<td>1 x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm Polystyrene Petri Dishes</td>
			<td>Fisherbrand</td>
			<td>0857512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light Dissection Microscope</td>
			<td>Motic</td>
			<td>SM2-168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Dissociation Materials</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube Rotator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td>Dissociation Tube Rotator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neural Tissue Dissociation Kit (P)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td>Enzymatic Dissociation Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>20-030-CV</td>
			<td>1 x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 &#956;m Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">A2B5 Column Purification&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-A2B5 Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-864</td>
			<td>Bead Bound A2B5 Antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Magnetic Selection Columns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Corning</td>
			<td>46-034-Cl</td>
			<td>2mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>20-030-CV</td>
			<td>1 x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td>0.50%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDGF-AA</td>
			<td>PeproTech Inc</td>
			<td>100-13A</td>
			<td>20 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Hybri-Max 100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triiodothyronine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6397</td>
			<td>45 nM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clemastine fumarate salt</td>
			<td>Sigma-Aldrich</td>
			<td>SML0445-100MG</td>
			<td>1 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quetiapine hemifumarate salt</td>
			<td>Sigma-Aldrich</td>
			<td>Q3638-10MG</td>
			<td>1 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td>5 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Progesterone</td>
			<td>Sigma-Aldrich</td>
			<td>P8783</td>
			<td>60 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1524</td>
			<td>10 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Putrecine</td>
			<td>Sigma-Aldrich</td>
			<td>P7505</td>
			<td>16 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Selenite</td>
			<td>Sigma-Aldrich</td>
			<td>S5261</td>
			<td>40 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0135</td>
			<td>50 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">d-Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td>10 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td>5 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Apo-Transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T1147</td>
			<td>100 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4161</td>
			<td>100 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trace Elements B</td>
			<td>Corning</td>
			<td>25-022-Cl</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td>1 x&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Black Visiplate with lid</td>
			<td>Perkin Elmer</td>
			<td>1450-605</td>
			<td>Tissue culture grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">Immunocytochemistry and Quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>100-13A</td>
			<td>4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>78787</td>
			<td>0.10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum</td>
			<td>Jackson Immuno Research</td>
			<td>005-000-121</td>
			<td>5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-MBP</td>
			<td>Biolegend</td>
			<td>SMI-99P</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit polyclonal anti-Actin</td>
			<td>Santa Cruz Biotecnology</td>
			<td>SC-7210</td>
			<td>1:200 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit polyclonal anti-Olig2</td>
			<td>Millipore</td>
			<td>AB9610</td>
			<td>1:1000 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse 680RD</td>
			<td>Licor</td>
			<td>926-68070</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit 800CW</td>
			<td>Licor</td>
			<td>926-32211</td>
			<td>1:500 Dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 goat anti-mouse</td>
			<td>Life Technologies</td>
			<td>A11032</td>
			<td>1:000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 goat anti-rabbit</td>
			<td>Life Technologies</td>
			<td>A11008</td>
			<td>1:000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold antifade with DAPI</td>
			<td>Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS with Ca2+ and Mg2+</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Licor Odyssey Clx Infared Imaging System</td>
			<td>Licor</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of rat oligodendrocyte progenitor cultures and quantification of oligodendrogenesis using dual-infrared fluorescence scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

A2B5-positive OPCs are isolated through positive cell selection using a magnetic column separation system. Prior to the isolation procedure, the whole brain is removed from rat pups that are between P5 and P7. Once the whole brain is successfully detached from the skull, the olfactory bulbs and cerebellum are removed using fine surgical forceps (Figure 1A). In order to isolate cerebral cortical tissue the hypothalamus, thalamus and midbrain are excised by careful dissecti...

Watch this Scientific Journal Video about Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning at JoVE.com

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

The overall goal of this procedure is to isolate and culture rat oligodendrocyte progenitor cells and quantify the oligodendrogenesis in response to experimental factors. Efficient oligodendrogenesis is the therapeutic goal in numerous areas of research, including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as transverse myelitis and multiple sclerosis. The main advantage of this technique is that it combines a protocol for the isolation of large numbers of OPCs with a fast and reliable method to determine oligodendrogenesis across multiple experimental conditions simultaneously.To begin this procedure, sterilize all the dissection equipment using a heated bead sterilizer. Then, add 15 to 20 milliliters of PBS to a 50 milliliter conical tube. Place the conical tube on ice, and use it to hold the diced cortex tissue during dissection.Next, add cold PBS to a 10 centimeter Petri dish which will be used for dissection under the light microscope. After sacrificing the rat pups, cut the skin behind the ears along the midline using small scissors. And peel the skin flaps back.Next, cut the cranium along the midline from the brain stem to the eyes. And be careful not to cut too deep in order to preserve the structure of the underlying cortex. After that, make two lateral cuts inferior to the cerebellum by inserting small surgical scissors into the foramen magnum.Make an additional cut between the eyes anterior to the olfactory bulbs. Carefully peel each half of the cranium back. Remove the whole brain and place it in a 10 centimeter Petri dish filled with cold PBS.Under a dissection light microscope, remove the olfactory bulbs and cerebellum with fine surgical forceps. Flip the brain so that the ventral surface is visible. Then, using fine straight forceps, perform a blunt dissection by placing closed forceps tips between the cortex and hypothalamus to a depth of about two-thirds of the brain.Open the forceps once they are in place, then repeat the procedure for the other hemisphere. After that, tease the cortex from the hypothalamus and midbrain regions. Remove the hypothalamus, thalamus, and midbrain by holding the midbrain at its posterior surface and peeling it toward the anterior end of the brain.Subsequently, sever the anterior connections. Using fine bent dissection forceps, remove the hippocampus and sever it's connection to the cortex. Then, remove the remaining striatum by scraping it from the underlying cortex in an outward diagonal motion.Clear the blood vessels and meninges from the ventral surface of the cortex. Next, flip the brain so that the dorsal surface is visible. Peel the meninges from the underlying cortex where the olfactory bulb is located.Repeat the procedures for a total of three to four rat pups. And place all the dissected cortices into a Petri dish. Chop them with a sterilized razor blade until one millimeter by one millimeter chunks are achieved.Then collect the tissues by rinsing the dish with PBS before placing it back on ice. Now, using a glass Pasteur pipette attached to a pipette controller, mechanically dissociate the tissue by pipetting ten to fifteen times or until the tissue pieces are reduced in size and the solution has become cloudy. Next, return the sample to the 37 degree Celsius tissue culture incubator for 15 minutes with continuous rotation.After that, add 10 milliliters of PBS to each sample and filter the homogenate through a 40 micrometer pore filter placed over a 50 milliliter tube. Then, rinse the filter with an additional one to two milliliters of PBS. Pool all the homogenate samples into one 50 milliliter conical tube and acquire an overall cell count.After performing the cell count, split the homogenate evenly into the 15 millileter conical tubes, and centrifuge them for 10 to 12 minutes at 300 gs. Prime each LS column by adding five milliliters of column buffer. And collect the flow through a 15 milliliter conical tube.Once the column buffer has completely passed through the upper chamber, apply 1000 microliters of the cell suspension to each column and allow the cells to run through by gravity. Next, remove each column and place it into a 15 milliliter conical tube. Add five milliliters of column buffer and plunge the buffer though the column at a fast rate to dislodge the bound cells and release them from the column.To plate the cells, dilute them with OPC proliferation media to 60, 000 cells per milliliter. Pipette 500 microliters of cells along the side of each black, clear bottom well of a 24 well plate. Mix the cells by shaking the plate horizontally, using a figure eight motion.In this procedure, place a 200 microliter pipette tip over a glass Pasteur pipette to ensure that the well is not damaged during aspiration. Then, aspirate the media from the culture wells one treatment group at a time to avoid drying the cells. After that, slowly add 500 microliters of treatment master mix along the side of the well using a one milliliter pipette.Incubate the cultures and perform a full media exchange with fresh treatment media every two to three days. At the end of the desired treatment period, aspirate the media. Slowly add 400 microliters of PFA to the side of the well to fix the cells.And incubate the plate for 20 minutes at room temperature. After 20 minutes, aspirate PFA and slowly add 500 microliters of sterile D-PBS to wash the cells. Repeat the wash two more times and do not aspirate the final wash.Now, bring the plate to room temperature, aspirate the wells, and add 250 microliters of D-PBS containing five percent NGS and 0.1 percent Triton x-100. Incubate the plate at room temperature for one hour with gentle orbital shaking. Prepare the primary incubation solution by diluting mouse monoclonal anti MBP antibody and rabbit polyclonal anti olig2 antibody in D-PBS containing five percent NGS.Then, incubate the primary antibodies at four degrees Celsius overnight for 16 to 18 hours with gentle orbital shaking. While washing the plate, prepare the secondary incubation solution by diluting the anti mouse 680, and anti rabbit 800 antibodies in D-PBS containing five percent NGS. Next, aspirate the final wash and add 250 microliters of secondary incubation solution.Protect the plate from light and incubate it at room temperature for one hour with gentle orbital shaking. After that, scan the plate using an imaging system capable of detecting 700 nanometer and 800 nanometer fluorescence emissions. As a starting point, set the focal offset to three, and sensitivities to one point five for MBP, five point zero for actin, and three point five for olig2.Adjust the sensitivity values as needed to avoid signal overexposure. In this schematic, the DIFS assay begins with freshly isolated A2B5 positive rat OPCs plated on PLL coded culture vessels. These cultures are proliferated for three days in the presence of 20 nanograms per milliliter PDGFAA, and then treated with experimental factors in fresh PDGF-free media on day zero.Treatment media is fully replenished on day two. And the cells are fixed with 4 percent paraformaldehyde on day four. Here, immunocytochemistry for olig2 and MBP was performed on the representative cultures on day zero and day four using Dapi as a nuclear counter stain.These cultures exhibit constitutive staining for the pan oligodendrocyte lineage cell marker, Olig2, on days zero and four. Whereas staining for mature oligodendrocyte marker MBP was only evident by day four. After watching this video you should have a good understanding of how to purify, culture and differentiate A2B5 positive rat OPCs in order to quantify the effect of experimental factors on oligodendrogenesis.The identification of factors that promote oligodenrogenesis is of particular relevance to the study of demyelinating diseases such as multiple sclerosis. Once mastered, the techniques described here may help to identify such factors and ultimately develop better therapeutics.

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Developmental Biology

11.6K Görüntüleme.  Johns Hopkins University, School of Medicine. The overall goal of this procedure is to isolate and culture rat oligodendrocyte progenitor cells and quantify the oligodendrogenesis in response to experimental factors. Efficient oligodendrogenesis is the therapeutic goal in numerous areas of research, including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as transverse myelitis and multiple sclerosis. The main advantage of this technique is that it combines a protocol for the isolation of large numbers of OPCs with...