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 water. Pipette 400 &#956;l of the diluted PLL into each well of a black-walled, clear bottom 24 well tissue culture grade dish.</li>
	<li>Coat tissue culture dishes for 2 hr in a 37 &#176;C tissue culture incubator or overnight in a 4 &#176;C refrigerator.</li>
	<li>Aspirate PLL from coated dishes at least 20 min prior to plating. Allow the remaining PLL to dry from the well by placing 24 well plates with the lid partially ajar in a tissue culture hood. Verify that the wells are completely dry before plating isolated OPCs. Cells will not adhere to plastic that has liquid PLL present.</li>
	<li>Prepare N-Acetyl-L-cysteine (NAC) stock solution (1,000x): Dissolve 100 mg N-Acetyl-L-cysteine (NAC) in 20 ml of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM). Aliquot and store at -20 &#176;C.</li>
	<li>Prepare Hydrocortisone stock solution (1,000x): Add 1 ml of ethanol to 1 mg Hydrocortisone and swirl to dissolve. Add 19 ml of DMEM, mix the solution, aliquot and store at -20 &#176;C. The addition of hydrocortisone to the base media has been shown to enhance the survival of glial cells14.</li>
	<li>Prepare d-Biotin stock solution (5,000x): Dissolve 2.5 mg of d-biotin in 50 ml of PBS. Add 1-2 x 5 &#181;l drops of 0.1 N NaOH to aid in dissolution. Aliquot and store at -20 &#176;C.</li>
	<li>Prepare Insulin stock solution (100x): Dissolve 25 mg Insulin in 50 ml of tissue culture grade water. Add 250 &#181;l of 1 N HCl and mix until solution is clear. Sterilize with a 0.22 &#181;m filter and store at 4 &#176;C for up to 6 weeks.</li>
	<li>Prepare Sato stock solution (100x):
	<ol>
		<li>Prepare progesterone stock solution (25 &#181;g/&#181;l): Dissolve 2.5 mg progesterone in 100 &#181;l of ethanol.</li>
		<li>Prepare sodium selenite stock solution (400 ng/&#181;l): Dissolve 4 mg sodium selenite in 100 &#181;l of 0.1 N NaOH. Add 10 ml of DMEM and mix.</li>
		<li>To 100 ml of DMEM, add 1 g of human apo-Transferrin, 1 g of bovine serum albumin, and 160 mg of putrescine and mix to fully dissolve. Add 25 &#181;l of progesterone stock solution, 1,000 &#181;l of sodium selenite stock solution, and mix. Aliquot and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare OPC Base Media: per 100 ml of DMEM (with 4.5 g/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate), add 100 &#181;l NAC, 100 &#181;l hydrocortisone, 20 &#181;l d-Biotin, 1 ml insulin, 1 ml Sato stock, 100 &#181;l trace elements B, 2 mL B-27 supplement (50x), and 1 ml of penicillin/streptomycin (100x). Sterilize with a 0.22 &#181;m filter and store at 4 &#176;C.</li>
	<li>Prepare PDGF-AA stock solution (1,000x): Dissolve 250 &#956;g in 12.5 ml of PBS with 0.1% bovine serum albumin (BSA) to make a 20 &#956;g/ml solution. Aliquot and store at -80 &#176;C.</li>
	<li>Prepare OPC proliferation media: OPC base media supplemented with 20 ng/ml PDGF-AA.</li>
	<li>Prepare OPC differentiation media: OPC base media supplemented with 45 nM triiodothyronine.</li>
	<li>Prepare Column Buffer: To 500 ml of PBS, add 2.5 g of BSA and 2 ml of 0.5 M ethylenediaminetetraacetic acid (EDTA) and adjust the pH to 7.2. Sterilize with a 0.22 &#181;m filter and store at 4 &#176;C.</li>
</ol>
2. Rat Brain Dissection
Note: P5-P7 rat pups are used in this protocol. Each rat pup cortex yields approximately 1.5-2.0 x 106 A2B5 positive cells.
<ol>
	<li>Sterilize all dissection equipment using a 250 &#176;C heated bead sterilizer.</li>
	<li>Add 15-20 ml of PBS (without Ca2+ and Mg2+) to 50 ml conical tubes. One 50 ml conical tube is sufficient for 3-4 rat pup cortices. Place 50 ml conical tubes on ice. Use 50 ml conical tubes to hold diced cortex tissue during dissection.</li>
	<li>Add cold PBS to a 10 cm Petri dish. This dish will be used for dissection under the light microscope.</li>
	<li>Sacrifice rat pups by decapitation with large surgical scissors. Spray head with 70% ethanol.</li>
	<li>Extract the Whole Brain
	<ol>
		<li>Using small scissors cut the skin down the midline and behind the ears and peel skin flaps back. Cut the cranium down the midline starting from the brainstem and ending at the eyes. Be careful not to cut too deeply in order to preserve the structure of the underlying cortex.</li>
		<li>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.</li>
		<li>Carefully peel each half of the cranium back. Remove the whole brain and place in the 10 cm Petri dish filled with cold PBS.</li>
	</ol>
	</li>
	<li>Under the dissection light microscope, remove the olfactory bulbs and cerebellum with fine surgical forceps.</li>
	<li>Flip the brain so that the ventral surface is visible under the light microscope.</li>
	<li>Using fine straight forceps perform a blunt dissection by placing closed forceps tips between the cortex and hypothalamus to a depth about 2/3 of the brain. Once forceps are in place allow the ends to open. Repeat for the other hemisphere.</li>
	<li>Tease the cortex from the hypothalamus and midbrain regions.</li>
	<li>Remove the hypothalamus, thalamus, and midbrain by holding the midbrain at its posterior surface and peeling it towards the anterior end of the brain. Sever the anterior connections.</li>
	<li>Remove the hippocampus by pealing it outward and then severing its connection to the cortex using fine bent dissection forceps.</li>
	<li>Remove remaining striatum by scrapping it from the underlying cortex in an outward diagonal motion using fine bent dissection forceps.</li>
	<li>Clear any blood vessels and meninges from the ventral surface of the cortex.</li>
	<li>Flip the brain so that the dorsal surface is visible. Meninges and blood vessels should be apparent. Peel meninges from the underlying cortex using fine dissection forceps. Start peeling from the olfactory bulb attachment location.</li>
	<li>Complete steps 2.4-2.14 for a total of 3-4 rat pups.</li>
	<li>Place 3-4 dissected rat pup cortices into a dry petri dish and chop with a sterilized razor blade until 1 mm x 1 mm chunks are achieved. Collect tissue by rinsing dish with PBS from one 50 ml conical tube (step 2) then place back on ice.</li>
</ol>
3. Enzymatic and Mechanical Tissue Dissociation
Note: For this step, a papain-based neural dissociation kit is recommended. Using Neural Dissociation Kit (P), special steps that are optimal for OPC isolation are described.
<ol>
	<li>Prepare the first dissociation enzyme by diluting enzyme P with the appropriate buffer. The contents of each 50 ml conical (1 sample) will be resuspended with a total of 1,950 &#181;l of enzyme mix. Place in the enzyme mix in a 37 &#176;C bath for 10 min. 
	&#160;&#160;&#160;&#160; 1 Sample:&#160;&#160; 1,900 &#181;l of Buffer + 50 &#181;l of papain enzyme</li>
	<li>Spin all 50 ml conical tubes containing 3-4 diced rat pup cortices at 300 x g for 3 min.</li>
	<li>Aspirate the supernatant and add 1,950 &#181;l of enzyme solution to each sample tube and break apart the pellet by inverting the tube and shaking gently.</li>
	<li>Incubate in a 37 &#176;C tissue culture incubator for 15 min with continuous tube rotation.</li>
	<li>During the last 5 min of the incubation, prepare the second enzyme mix A. Dilute the enzyme in its appropriate buffer. A total of 30 &#181;l will be added to each 50 ml conical sample tube. 
	&#160;&#160;&#160;&#160; 1 Sample:&#160;&#160; 20 &#181;l of Buffer + 10 &#181;l of enzyme</li>
	<li>Once incubation is completed add 30 &#181;l of the second enzyme mix to each tube and invert to mix.</li>
	<li>Using a glass Pasteur pipette attached to a pipette controller, mechanically dissociate the tissue by pipetting 10-15 times or until the tissue pieces are reduced in size and the solution has become cloudy. Return the sample to the 37 &#176;C tissue culture incubator for 15 min with continuous rotation.</li>
	<li>Pipette each tissue homogenate sample 10-15 times using a 1 ml pipette.</li>
	<li>Pipette each tissue homogenate sample 15-20 times using a 200 &#181;l pipette</li>
	<li>Return all samples to the 37 &#176;C tissue culture incubator for 10 min with continuous rotation.</li>
	<li>Add 10 ml of PBS (with Ca2+ and Mg2+) to each sample and filter the homogenate through a 40 &#181;m pore filter placed over a 50 ml tube. Rinse the filter with an additional 1-2 ml of PBS.</li>
	<li>Pool all of the homogenate samples into one 50 ml conical tube and acquire an overall cell count.</li>
	<li>After performing the cell count, split the homogenate evenly into 15 ml conical tubes (1 tube for each sample). Centrifuge for 10-12 min at 300 x g.</li>
</ol>
4. Anti-A2B5 Bead Application, Column Purification, and Plating
<ol>
	<li>Perform calculations for column buffer and anti-A2B5 microbeads. Add 7 &#956;l Column Buffer for every 1 x 106 cells. Add 2 &#956;l Anti-A2B5 microbeads for every 1 x 106 cells.</li>
	<li>Combine all of the samples by resuspending the cells in a total volume of 10 ml of column buffer and centrifuge at 300 x g for 10-12 min.</li>
	<li>Resuspend the cell pellet in the appropriate amount of column buffer as calculated in step 4.1. If the pellet is large and loose, only add approximately half of the volume of column buffer to keep the antibody at the appropriate concentration in the cell resuspension.</li>
	<li>Incubate the cell suspension for 10 min at 4 &#176;C.</li>
	<li>Add anti-A2B5 microbeads (calculated in step 4.1), invert several times and incubate at 4 &#176;C for 30-60 min. Gently invert the tube several times every 10 min. Longer incubation times may increase cell yields but may increase non-specific binding.</li>
	<li>Add 5 volumes of column buffer. If the cells are resuspended in a volume of 1 ml, add 5 ml of column buffer, whereas if the cells are resuspended in 2 ml, add 10 ml of column buffer.</li>
	<li>Centrifuge for 10 min at 300 x g.</li>
	<li>Determine how many LS columns will be needed for the purification. One LS column is sufficient for 15-20 x 106 total cells.</li>
	<li>Resuspend the cell pellet in 1,000 &#181;l of column buffer per LS column being used.</li>
	<li>Prime each LS column by adding 5 ml of column buffer. Collect the flow through in a 50 ml conical tube. Once the column buffer has completely passed through the upper chamber, apply 1,000 &#181;l of the cell suspension to each column and allow the cells to run through by gravity. 
	Note: If the density of cells is too high, the column may run slow.</li>
	<li>Immediately after the cell suspension has passed through the column, slowly add 3 ml of column buffer to each column to wash the unbound cells. If the wash is readily running through the column (1 drop for every 30-45 sec), wash two additional times with 3 ml of column buffer.</li>
	<li>Remove each column and place it on a 15 ml conical tube. Add 5 ml of column buffer and plunge the buffer through the column at a fast rate using the plastic plunger that is packaged with the column. Plunging will dislodge the bound cells releasing them from the column.</li>
	<li>Increase purity by running the sample through a second round of LS columns. Use one third of the number of columns used during the first pass. Prime the columns with 5 ml of column buffer. Allow the column buffer to pass through the upper chamber.</li>
	<li>Since the volume of cell suspension will be much greater, evenly apply 1,000 &#181;l of cell suspension to each column and allow the suspension to pass through the upper chamber of the column before replenishing with an additional 1,000 &#181;l.</li>
	<li>Once the cell suspension has been completely applied to the second round of columns, wash 2-3 times with 3 ml of column buffer.</li>
	<li>Remove each column and place it over a sterile 15 ml conical tube. Add 5 ml of column buffer and plunge the buffer using the plastic plunger that is packaged with the column. Plunging will dislodge the bound cells releasing them from the column.</li>
	<li>Centrifuge the cell suspension at 300 x g for 12-15 min. The pellet should appear compact.</li>
	<li>Resuspend the cell pellet in 5-10 ml of pre-warmed OPC proliferation media (OPC base media supplemented with PDGF-AA 20 ng/ml).</li>
	<li>Count the cells using a hemocytometer.</li>
	<li>Plate the cells. Dilute the cells to 60,000 cells/ml with OPC proliferation media. Into each black, clear bottom 24 well, pipet 500 &#181;l of cells along the side of the well to obtain 30,000 cells per well. Mix the cells by shaking the plate horizontally using a figure eight motion. If performing the assay in 48, 96, or 384-well plates, add 15,000, 7,500, or 1,500 cells per well, respectively. These numbers may be adjusted depending on individual plate specifications.</li>
	<li>Prepare a separate plate for the Day 0 baseline control cultures. This control plate should be fixed with 4% paraformaldehyde as described in section 5.7, when differentiation is initiated on Day 0.</li>
	<li>Culture the cells in OPC proliferation media at 37 &#176;C for 3 days with no media change.</li>
</ol>
5. Induction of OPC Differentiation and Fixation with 4% Paraformaldehyde
Note: When testing the effect of small molecules on oligodendrogenesis, we routinely dissolve drugs in dimethyl sulfoxide (DMSO) to prepare 1,000x stocks that can then be stored at -80 &#176;C and diluted directly into SATO media to obtain a final concentration of 0.1% DMSO. We have observed no changes in either OPC proliferation or differentiation using this concentration of DMSO (data not shown).
<ol>
	<li>Pre-warm OPC base media to 37 &#176;C and thaw drug treatment stocks to room temperature. When performing treatments in duplicate, prepare approximately 1.25 ml of media per treatment group in a 1.5 ml tube. For triplicate samples, use 2 ml capacity centrifuge tubes to account for slack.</li>
	<li>Prepare the treatment master mixes for replicate wells by diluting treatment stocks directly into OPC base media. Briefly vortex or gently mix each mastermix to ensure homogeneous distribution of the treatment.</li>
	<li>Prepare 45 nM triiodothyronine (T3) as the positive control and the appropriate vehicle as the negative control. Dilute 10 mM T3 stock 1:1,000 in 1 ml of OPC base media and then further dilute in the treatment master mix to reduce the final concentration of DMSO if necessary.</li>
	<li>Aspirate the media from the culture wells one treatment group at a time so as to avoid drying the cells. Use a 200 &#181;l pipet tip on the end of the glass Pasteur pipet to avoid scraping off black material from the sides of the well and into the culture.</li>
	<li>Slowly add 500 &#181;l of treatment mastermix along the side of the well using a 1 ml pipet.</li>
	<li>Incubate the cultures for the desired timeframe, performing a full media exchange with fresh treatment media every 2-3 days. We routinely observe a 30-40% MBP+ culture after 4 days in differentiation media.</li>
	<li>At the end of the desired treatment period, prepare fresh 4% paraformaldehyde (PFA) or thaw a frozen stock to room temperature. CAUTION: PFA is extremely toxic and must be prepared in a fume hood.</li>
	<li>Aspirate the media and slowly add 400 &#181;l of PFA to the side of the well to fix the cells. Incubate the plate for 20 min at room temperature.</li>
	<li>Aspirate the PFA and slowly add 500 &#181;l of sterile D-PBS (with Ca2+ and Mg2+) to the side of the well to wash the cells. Repeat the wash a total of two more times. Do not aspirate the final wash.</li>
	<li>Wrap the plate in parafilm and store at 4 &#176;C for later processing or proceed directly to the next step. Plates can be stored for several weeks.</li>
</ol>
6. Immunocytochemistry and Quantification of Myelin Basic Protein
Note: When performing liquid handling procedures in the 24 well plates, it is recommended to work fast to avoid drying the cells. Here, the use of a multi-channel vacuum aspirator and multi-channel pipet are recommended.
<ol>
	<li>Bring the plate to room temperature, aspirate the wells, and add 250 &#956;l of D-PBS (with Ca2+ and Mg2+) containing 0.1% Triton X-100 and 5% normal goat serum (NGS). Incubate the plate at room temperature for 1 hr with gentle orbital shaking.</li>
	<li>Prepare the primary incubation solution by diluting mouse monoclonal anti-MBP antibody 1:1,000 and rabbit polyclonal anti-Actin antibody 1:200 in D-PBS (with Ca2+ and Mg2+) containing 5% NGS. Alternatively, rabbit polyclonal anti-Olig2 at 1:1,000 can be used for oligodendrocyte specific normalization in mixed glial cultures. Prepare enough primary solution to accommodate 250 &#181;l per well.</li>
	<li>Incubate primary antibodies at 4 &#176;C overnight for 16-18 hr with gentle orbital shaking.</li>
	<li>Aspirate the primary incubation solution and wash the cells 3 x 10 min with 500 &#181;l of D-PBS (with Ca2+ and Mg2+) at room temperature with gentle orbital shaking.</li>
	<li>While washing the plate, prepare the secondary incubation solution by diluting anti-mouse 680 and anti-rabbit 800 antibodies 1:500 in D-PBS (with Ca2+ and Mg2+) containing 5% NGS. Minimize ambient light exposure when preparing this solution.</li>
	<li>Aspirate the final wash and add 250 &#181;l of secondary incubation solution. Protect the plate from light and incubate it at room temperature for 1 hr with gentle orbital shaking.</li>
	<li>Aspirate the secondary incubation solution and wash the wells 3 x 10 min with 500 &#181;l of D-PBS (with Ca2+ and Mg2+) at room temperature with gentle orbital shaking.</li>
	<li>Scan the plate using an imaging system capable of detecting 700 nm and 800 nm fluorescence emissions. As a starting point, set the focal offset to 3 and sensitivities to 1.5 for MBP, 5.0 for Actin, and 3.5 for Olig2. Adjust the sensitivity values as needed to avoid signal overexposure.</li>
	<li>Quantify the extent of oligodendrogenesis using semi-automated software-based methods.
	<ol>
		<li>Using the software, set the plate analysis mode to &#34;24 Well&#34; and align circles around the outer perimeter of the scanned wells. Set the normalization channel to &#34;800&#34; and export the total and relative fluorescence intensities for the 700 nm (MBP) and 800 nm (Olig2/Actin) channels.</li>
		<li>Divide the intensity at 700 nm by the intensity at 800 nm to give the normalized MBP expression in relative fluorescence units. Calculate the average of replicate measurements and scale the expression to the Day 0 +PDGF controls as needed for the analysis.</li>
	</ol>
	</li>
</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=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><tbody><tr><td>&lt;strong&gt;Dissection Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Mediatech</td><td>21-040-CV</td><td>1x</td></tr><tr><td>10 cm Polystyrene Petri Dishes</td><td>Fisherbrand</td><td>0857512</td><td></td></tr><tr><td>Light Dissection Microscope</td><td>Motic</td><td>SM2-168</td><td></td></tr><tr><td>&lt;strong&gt;Dissociation Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tube Rotator</td><td>Miltenyi Biotec</td><td>130-090-753</td><td>Dissociation Tube Rotator</td></tr><tr><td>Neural Tissue Dissociation Kit (P)</td><td>Miltenyi Biotec</td><td>130-092-628</td><td>Enzymatic Dissociation Kit</td></tr><tr><td>PBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Corning</td><td>20-030-CV</td><td>1x</td></tr><tr><td>40 &amp;mu;m Cell Strainer</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>&lt;strong&gt;A2B5 Column Purification&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-A2B5 Microbeads</td><td>Miltenyi Biotec</td><td>130-097-864</td><td>Bead Bound A2B5 Antibody</td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>Magnetic Selection Columns</td></tr><tr><td>EDTA</td><td>Corning</td><td>46-034-Cl</td><td>2 mM</td></tr><tr><td>PBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Corning</td><td>20-030-CV</td><td>1x</td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9647</td><td>0.50%</td></tr><tr><td>&lt;strong&gt;Culture Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PDGF-AA</td><td>PeproTech Inc</td><td>100-13A</td><td>20 ng/ml</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D2650</td><td>Hybri-Max 100 ml</td></tr><tr><td>Triiodothyronine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6397</td><td>45 nM</td></tr><tr><td>Clemastine fumarate salt</td><td>Sigma-Aldrich</td><td>SML0445-100MG</td><td>1 &amp;mu;M</td></tr><tr><td>Quetiapine hemifumarate salt</td><td>Sigma-Aldrich</td><td>Q3638-10MG</td><td>1 &amp;mu;M</td></tr><tr><td>N-acetyl cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td>5 &amp;mu;g/ml</td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P8783</td><td>60 ng/ml</td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P1524</td><td>10 &amp;mu;g/ml</td></tr><tr><td>Putrecine</td><td>Sigma-Aldrich</td><td>P7505</td><td>16 &amp;mu;g/ml</td></tr><tr><td>Sodium Selenite</td><td>Sigma-Aldrich</td><td>S5261</td><td>40 ng/ml</td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0135</td><td>50 ng/ml</td></tr><tr><td>d-Biotin</td><td>Sigma-Aldrich</td><td>B4639</td><td>10 ng/ml</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td>5 &amp;mu;g/ml</td></tr><tr><td>Apo-Transferrin</td><td>Sigma-Aldrich</td><td>T1147</td><td>100 &amp;mu;g/ml</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A4161</td><td>100 &amp;mu;g/ml</td></tr><tr><td>Trace Elements B</td><td>Corning</td><td>25-022-Cl</td><td>1x&amp;nbsp;</td></tr><tr><td>B-27 Supplement</td><td>Life Technologies</td><td>17504044</td><td>1x&amp;nbsp;</td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15140122</td><td>1x&amp;nbsp;</td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11995-065</td><td>1x&amp;nbsp;</td></tr><tr><td>24-well Black Visiplate with lid</td><td>Perkin Elmer</td><td>1450-605</td><td>Tissue culture grade</td></tr><tr><td>&lt;strong&gt;Immunocytochemistry and Quantification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PFA</td><td>Sigma-Aldrich</td><td>100-13A</td><td>4%</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>78787</td><td>0.10%</td></tr><tr><td>Normal Goat Serum</td><td>Jackson Immuno Research</td><td>005-000-121</td><td>5%</td></tr><tr><td>mouse monoclonal anti-MBP</td><td>Biolegend</td><td>SMI-99P</td><td>1:1,000 Dilution</td></tr><tr><td>rabbit polyclonal anti-Actin</td><td>Santa Cruz Biotecnology</td><td>SC-7210</td><td>1:200 Dilution</td></tr><tr><td>rabbit polyclonal anti-Olig2</td><td>Millipore</td><td>AB9610</td><td>1:1,000 Dilution</td></tr><tr><td>goat anti-mouse 680RD</td><td>Licor</td><td>926-68070</td><td>1:500 Dilution</td></tr><tr><td>goat anti-rabbit 800CW</td><td>Licor</td><td>926-32211</td><td>1:500 Dilution</td></tr><tr><td>Alexa Fluor 594 goat anti-mouse</td><td>Life Technologies</td><td>A11032</td><td>1:1,000 dilution</td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit</td><td>Life Technologies</td><td>A11008</td><td>1:1,000 dilution</td></tr><tr><td>Prolong Gold antifade with DAPI</td><td>Life Technologies</td><td>P36931</td><td></td></tr><tr><td>DPBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>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 ...

preparation-rat-oligodendrocyte-progenitor-cultures-quantification

Research

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Developmental Biology

11.8K Views.  Johns Hopkins University, School of Medicine. 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.

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

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Neuroscience

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Rat "Brain Milking" for Neural Stem and Oligodendrocyte Progenitor Cell Isolation

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Author Spotlight: Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

Dual Fluorescence Imaging to Identify Differentiated Oligodendrocytes

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/t/53764/preparation-of-rat-oligodendrocyte-progenitor-cultures-and-quantification-of-oligodendrogenesis-using-dual-infrared-fluorescence-scanning'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Schott, J. T.,&#160;et al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2016).</a> &#160;This video demonstrates the use of dual fluorescence imaging to evaluate the differentiation of oligodendrocytes from precursor cells (OPCs) isolated from a rat pup brain. The process includes fixing the cells, labeling specific markers with primary and fluorophore-conjugated secondary antibodies, and visualizing them using fluorescence imaging. Differentiated oligodendrocytes expressing both myelin basic protein (MBP) and oligodendrocyte transcription factor 2 (Olig2) are identified, distinguishing them from undifferentiated precursor cells that express only Olig2.

Source:&#160;Schott, J. T.,&#160;et al. Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using ...

JoVE Encyclopedia of Experiments

Neuroimaging

Structural & Functional Brain Imaging

Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

Source: Portela-Lomba, M., et al. <a href="https://app.jove.com/v/61863">Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to isolate retinal ganglion neurons (RGNs) from a rat&#39;s retina and coculture them with olfactory ensheathing glia (OEG) cells. This method allows for studying the neuro-regenerative potential of OEGs after neural injury.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

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