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

Grant patrocinador: Instituto Nacional de Salud; Grant número: R37 NS041435.

Los procedimientos que implican sujetos animales han sido aprobados por Cuidado de Animales institucional y el empleo Comisión (IACUC) y Johns Hopkins School of Medicine. 1. Preparación de placas de ensayo, solución madre, y OPC base para medios Nota: La base de rata (OPC) Sato medios descritos aquí viene de los estudios previamente publicados 12,13. formulaciones de medios alternativos también pueden ser compatibles con este procedimiento. <ol><li> Resuspender poli-L-lisina (PLL) a una...

<ol>
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El protocolo que se presenta aquí describe un método rápido y fiable para el aislamiento de los OPC de rata y cuantificar en oligodendrogenesis vitro en respuesta a factores experimentales. Usando la selección positiva, altos rendimientos de los OPC viables y puros se utilizan directamente para fines experimentales. Este método simplifica el aislamiento, cultivo y etapas de diferenciación en un marco de tiempo 1 semana, y las células fijadas se puede almacenar convenientemente a 4 ° C durante v...

conducción nerviosa eficiente en el sistema nervioso central de los mamíferos (CNS) requiere la mielinización de los axones por oligodendrocitos. Durante el desarrollo, surgen los oligodendrocitos a partir de un conjunto de células progenitoras de oligodendrocitos (OPC) que se cree que la migración de las zonas ventriculares del cerebro anterior en desarrollo y el tubo neural 1. Después de los OPC de migración se diferencian en oligodendrocitos maduros, myelinating que no sólo facilitar la propagación del impulso eficiente, sino que también proporcionan los axones con soporte trófico 2. El sistema nervioso central adulto mantiene una abundante población de los OPC que están dispersos en toda la sustancia gris y blanco que comprende aproximadamente el 5-8% de todas las células 3. Luego de una lesión desmielinizante, los OPC migran al sitio de la lesión, proliferan y se diferencian para sustituir a las vainas de mielina perdidos o dañados en los axones expuestos. Sin embargo, en algunos lugares la enfermedad / lesión, este proceso se encuentra para ser ineficaz o puede fallar por completo 4. Mientrasdesmielinización crónica se cree que añadir a la carga de morbilidad, la remielinización oligodendrogenesis y eficiente puede aliviar los síntomas 5. Por ello, ha sido de interés para estudiar los OPC in vitro para determinar el efecto de los factores experimentales sobre oligodendrogenesis. La comprensión de las diferentes fases de la diferenciación de oligodendrocitos ha sido posible gracias a la identificación de marcadores específicos de células de la etapa. Auto-renovación de células progenitoras tempranas se definen por su expresión de alfa derivado de plaquetas receptor del factor de crecimiento (PDGFR), el antígeno neuronal / glía 2 (NG2) y A2B5 6 - 8. Como OPCs inician su programa de diferenciación y se retiran del ciclo celular, que regulan negativamente la expresión de estos marcadores y comienzan a expresar proteínas indicativas de premielinizantes oligodendrocitos incluyendo cíclico de nucleótidos 3&#39;-fosfodiesterasa (CNPase) y O4. Por último, su diferenciación en oligodendroc más maduroytes se caracteriza por la expresión de proteínas asociados a la mielina, incluyendo la glicoproteína asociada a la mielina (MAG), proteína proteolipídica (PLP), y la proteína básica de la mielina (MBP). MBP es una proteína de membrana periférica intracelular y un componente principal de la vaina de mielina. Los ratones que carecen MBP desarrollan un fenotipo grave en el que la mielinización del SNC está disminuida significativamente dando lugar a temblores, convulsiones y muerte temprana 9,10. Este importante papel de la MBP en la mielinización ha llevado a su uso como un marcador de la diferenciación de oligodendrocitos in vitro e in vivo 11. La cuantificación de MBP se puede lograr usando varias metodologías diferentes. RT-PCR y análisis de transferencia Western permiten la cuantificación de los niveles de MBP bajo diferentes regímenes de tratamiento. La inmunocitoquímica es un enfoque cualitativo común que también puede ser cuantitativa cuando se usan enfoques de microscopía basados ​​en cámaras. Aunque estos sistemas son fiables y fundamental parael estudio de la diferenciación de oligodendrocitos, cada uno de ellos tiene sus propias desventajas que limitan su uso en la detección de drogas. En primer lugar, la cantidad de OPCs principales que se necesitan para RT-PCR y análisis de transferencia Western reduce el número de variables que se pueden examinar simultáneamente. Mientras que el requisito para inmunocitoquímica de células es mucho más bajo, un tiempo considerable se debe dedicar a cada experimento si la cuantificación es la meta. Numerosas imágenes deben ser capturados y luego cuantificados para facilitar el análisis experimental. Estas advertencias se convierten en obstáculos importantes a tener en cuenta para los estudios que requieren una evaluación de alto rendimiento. Aquí se describe un método que utiliza aspectos fundamentales tanto de la inmunocitoquímica y metodologías de Western blot para la cuantificación de la mielina, mientras que reduce significativamente tanto el número de células requeridas y el tiempo para completar el análisis cuantitativo.

<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-positivas se aislaron mediante selección celular positiva utilizando un sistema de separación magnética de columna. Antes del procedimiento de aislamiento, todo el cerebro se elimina de crías de rata que se encuentran entre P5 y P7. Una vez que todo el cerebro se separa con éxito desde el cráneo, los bulbos olfatorios y el cerebelo se eliminan mediante unas pinzas quirúrgicas finas (Figura 1A). Con el fin de aislar el tejido cortical cerebral el hipotála...

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.

Preparación de la rata progenitoras de oligodendrocitos culturas y cuantificación de Oligodendrogenesis Uso del análisis de fluorescencia de doble infrarrojo

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

Culturas organotípicos rebanada de Estudios de Postnatal neurogénesis

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

Investigación

Biología del Desarrollo

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

Entrega de<em&gt; En Vivo</em&gt; Aguda intermitente hipoxia neonatal en roedores a derivados de la Zona Prime subventricular progenitoras neurales Cultivos Celulares

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

Aislamiento de células neurales madre / progenitoras de la región periventricular de la rata de Adultos y de la médula espinal humana

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

Generación de Genéticamente Modificados cultivos de piel organotípicos Uso Desvitalizada Dermis Humana

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

El uso de cultivos de neuroesferas primarias estudian primaria cilios

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

Sola célula cuantificación de las tasas de degradación de proteína por microscopia de fluorescencia Time-lapse en cultivo de células adherentes

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

Visualización de la placa notocordal en embriones de ratón Gastrulating con microscopía electrónica e inmunofluorescencia de Monte todo y nodo

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 de ensayo de angiogénesis tridimensional utilizando esferoides de cocultura formados por células formadoras de colonias endoteliales y células madre mesenquimales

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

Análisis del metabolismo de células hematopoyéticas del progenitor del tallo

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

Cuantificación de la auto-renovación en las culturas de la maméfera de la muriña

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