Name: preparation of rat oligodendrocyte progenitor cultures and quantification of oligodendrogenesis using dual-infrared fluorescence scanning
Uploaded: 2016-02-17T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning at JoVE.com

Concedere sponsor: National Institutes of Health; codice di autorizzazione: R37 NS041435.

Le procedure che coinvolgono soggetti animali sono stati approvati dalla Institutional Animal Care e Usa Comitato (IACUC) e la Johns Hopkins School of Medicine. 1. Preparazione di piatti analisi, soluzioni di riserva, e OPC base per supporti multimediali Nota: La base di ratto OPC (Sato) i media qui descritte sono state derivate da studi pubblicati in precedenza 12,13. formulazioni dei media alternativi possono anche essere compatibili con questa procedura. <ol><li> Risospendere poli-L-lisin...

<ol>
	<li>Rowitch, D. H., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+vertebrate+glial-cell+specification.">Developmental genetics of vertebrate glial-cell specification.</a> Nature. 468 (7321), 214-222 (2010).</li><li>Nave, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+and+the+trophic+support+of+long+axons.">Myelination and the trophic support of long axons.</a> Nat Rev Neurosci. 11 (4), 275-283 (2010).</li><li>Dawson, M. R. L., Polito, A., Levine, J. M., Reynolds, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-expressing+glial+progenitor+cells:+an+abundant+and+widespread+population+of+cycling+cells+in+the+adult+rat+CNS.">NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.</a> Mol Cell Neurosci. 24 (2), 476-488 (2003).</li><li>Goldschmidt, T., Antel, J., K&#246;nig, F. B., Br&#252;ck, W., Kuhlmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remyelination+capacity+of+the+MS+brain+decreases+with+disease+chronicity.">Remyelination capacity of the MS brain decreases with disease chronicity.</a> Neurology. 72 (22), 1914-1921 (2009).</li><li>Duncan, I. D., Brower, A., Kondo, Y., Curlee, J. F., Schultz, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+remyelination+of+the+CNS+leads+to+functional+recovery.">Extensive remyelination of the CNS leads to functional recovery.</a> Proc Natl Acad Sci USA. 106 (16), 6832-6836 (2009).</li><li>Nishiyama, A., Chang, A., Trapp, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2++glial+cells:+a+novel+glial+cell+population+in+the+adult+brain.">NG2+ glial cells: a novel glial cell population in the adult brain.</a> J Neuropath Exp Neur. 58 (11), 1113-1124 (1999).</li><li>Baracskay, K. L., Kidd, G. J., Miller, R. H., Trapp, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-positive+cells+generate+A2B5-positive+oligodendrocyte+precursor+cells.">NG2-positive cells generate A2B5-positive oligodendrocyte precursor cells.</a> Glia. 55 (10), 1001-1010 (2007).</li><li>Ffrench-Constant, C., Raff, M. 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>

Il protocollo presentato qui descrive un metodo veloce e affidabile per isolare OPC ratto e quantificare in oligodendrogenesis vitro in risposta a fattori sperimentali. Utilizzando selezione positiva, alte rese di OPC vitali e puri sono utilizzati direttamente a fini sperimentali. Questo metodo semplifica l&#39;isolamento, la coltura, e passi differenziazione in un arco di tempo 1 Settimana, e cellule fissate possono essere convenientemente conservato a 4 ° C per diverse settimane senza alcuna perdita...

Efficiente conduzione nervosa nel sistema nervoso centrale dei mammiferi (CNS) richiede mielinizzazione degli assoni dagli oligodendrociti. Durante lo sviluppo, oligodendrociti nascono da un pool di cellule progenitrici degli oligodendrociti (OPC) che si pensa di migrare dalle zone ventricolari del prosencefalo sviluppo e tubo neurale 1. Dopo OPC migrazione differenziano in maturi, oligodendrociti mielinizzanti che non solo facilitare l&#39;utilizzo efficiente di propagazione degli impulsi, ma anche fornire gli assoni con il supporto trofico 2. L&#39;adulto CNS mantiene una popolazione abbondante di OPC che si disperdono in tutta la materia grigia e bianca che comprende circa il 5-8% di tutte le cellule 3. A seguito di un insulto demielinizzante, OPC migrano al sito di lesione, proliferano e si differenziano per sostituire guaine mieliniche persi o danneggiati in assoni esposti. Tuttavia, in alcune impostazioni malattia / infortunio, questo processo si trova ad essere inefficiente o può fallire completamente 4. Mentredemielinizzazione cronica è pensato di aggiungere al peso della malattia, oligodendrogenesis e rimielinizzazione efficiente può alleviare i sintomi 5. È stato quindi interessante studiare OPC in vitro per determinare l&#39;effetto di fattori sperimentali oligodendrogenesis. Insight nelle diverse fasi di differenziazione degli oligodendrociti è stato reso possibile tramite l&#39;identificazione di marcatori cellulari specifici stadi. Auto-rinnovamento delle cellule progenitrici precoci sono definite dalla loro espressione del fattore di crescita derivato dalle piastrine del recettore alfa (PDGFRα), neurale / glia antigene 2 (NG2) e A2B5 6-8. Come OPC iniziano il loro programma di differenziazione e si ritirano dal ciclo cellulare, che downregulate espressione di questi marcatori e cominciano a esprimere le proteine ​​indicativi di premyelinating oligodendrociti tra ciclico-nucleotide 3&#39;-fosfodiesterasi (CNPase) e O4. Infine, la loro differenziazione in oligodendroc più maturoYtes si caratterizza per l&#39;espressione di proteine ​​della mielina-associata, tra cui glicoproteina mielina-associata (MAG), proteolipid proteina (PLP), e proteina basica della mielina (MBP). MBP è una proteina periferica di membrana intracellulare e una componente importante della guaina mielinica. Topi privi MBP sviluppano una grave fenotipo in cui CNS mielinizzazione è significativamente diminuiti con conseguente tremori, convulsioni e morte precoce 9,10. Questo ruolo importante MBP in myelination ha portato al suo uso come marker di differenziazione degli oligodendrociti sia in vitro che in vivo 11. La quantificazione del MBP può essere effettuata mediante diverse metodologie differenti. RT-PCR e analisi Western Blot consentono per la quantificazione dei livelli di MBP sotto diversi regimi di trattamento. Immunocytochemistry è un approccio qualitativo comune che può anche essere quantitativa quando si utilizzano approcci microscopia basati su telecamere. Sebbene questi sistemi sono affidabili e fondamentale perlo studio della differenziazione degli oligodendrociti, ognuno ha i propri svantaggi che limitano il loro uso in screening di stupefacenti. In primo luogo, la quantità di OPC primari che sono necessari per la RT-PCR ed analisi Western blot riduce il numero di variabili che possono essere esaminati simultaneamente. Mentre il requisito cella per immunocitochimica è molto più basso, consistente lasso di tempo deve essere dedicato a ogni esperimento, se la quantificazione è l&#39;obiettivo. Numerose immagini devono essere catturate e poi quantificati per facilitare l&#39;analisi sperimentale. Questi avvertimenti diventano ostacoli importanti da considerare per gli studi che richiedono la valutazione elevato throughput. Qui si descrive un metodo che utilizza aspetti fondamentali sia dal immunocitochimica e metodologie Western Blot per mielina quantificazione, riducendo significativamente sia il numero di cellule necessario e il tempo per completare l&#39;analisi quantitativa.

<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.

OPC A2B5-positivi vengono isolati attraverso la selezione delle cellule positivo utilizzando un sistema di separazione della colonna magnetica. Prima della procedura di isolamento, l&#39;intero cervello viene rimosso da cuccioli di ratto che sono tra P5 e P7. Una volta che l&#39;intero cervello si stacca con successo dal cranio, bulbi olfattivi e del cervelletto vengono rimossi con pinza chirurgica sottili (Figura 1A). Per isolare tessuto corticale cerebrale ipotalamo, t...

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.

Preparazione delle Culture Rat progenitrici degli oligodendrociti e la quantificazione dei Oligodendrogenesis Utilizzando Dual-infrarossi fluorescenza Scansione

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

Research

JoVE Journal

Developmental Biology

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Colture organotipiche Slice di studi di post-natale neurogenesi

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Ricerca

Biologia dello sviluppo

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Consegna di<em&gt; In Vivo</em&gt; Acuta intermittente ipossia in Neonatale Roditore al Primo subventricolare Zone-derivati ​​colture cellulari progenitrici neurali

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Isolamento di Neural cellule staminali / progenitrici dalla Regione periventricolare del ratto adulto e midollo spinale umano

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Generazione di culture geneticamente modificate pelle organotipiche Uso devitalizzate derma umano

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Using Primary Neurosphere Cultures to Study Primary Cilia

Utilizzando colture di neurosfere primarie al primario dello studio Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Cella singola quantificazione dei tassi di degradazione delle proteine mediante microscopia a fluorescenza time-lapse in coltura delle cellule aderenti

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Visualizzare il nodo e la piastra Notochordal In embrioni di topo Gastrulating mediante microscopia elettronica a scansione e intero Monte immunofluorescenza

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Sistema di analisi dell'angiogenesi tridimensionale utilizzando Gli spheroidi co-culturali formati da cellule formanti colonia endoteliale e cellule staminali mesenchiche

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Analisi del metabolismo delle cellule staminali ematopoietiche

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

Quantification of Self-renewal in Murine Mammosphere Cultures

Quantificazione dell'autorinnovamento nelle colture di Mammosfera Murine

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...