Para trabajar en condiciones estériles, se recomienda llevar a cabo todos los pasos en una campana de flujo laminar. 1. Materiales <ul><li> La disociación del tejido neural Kit (P) (Componentes: Solución de 1,2, 3 y 4, del buffer de almacenamiento) </li><li> Separación de los pre-filtros </li><li> (Opcional) La mielina bolas de eliminación </li><li> Beta-mercaptoetanol </li><li> gentleMACS disociador </li><li> C Tubos </li><li> MACSmix rotador del tubo </li><li> HBSS (w / o) </li><li> HBSS (w) </li></ul><ol><li> Prepare las siguientes soluciones antes de iniciar el protocolo: <ol><li> Buffered Hanks Solución salina sin cationes divalentes Ca 2 + o Mg 2 + (HBSS (w / o) </li><li> HBSS, estándar, es decir, con Ca 2 + y Mg 2 + (HBSS (w) </li></ol></li><li> Dependiendo del epítopo del antígeno de interés en las aplicaciones posteriores, utilice la papaína basada en tejidos neuronales disociación Kit (P) o la tripsina basada en tejidos neuronales disociación Kit (T). </li><li> Prepare las siguientes soluciones de Neural tejidos disociación del kit: <ol><li> Solución 2: añadir beta-mercaptoetanol al 0,067 mM </li><li> Solución 4: disolver el polvo en 0,7 ml de Buffer de almacenamiento (incluye en el kit), mezcle suavemente (no vortex) </li><li> Mezcle una enzima: Pipetear 1,9 ml de la Solución 2 y 50 l Solución 1 (tanto de equipo) en un tubo C e incubar durante 10-15 min a 37 ° C. Esto es suficiente para disociar 400 mg de tejido cerebral. </li></ol></li></ol> 2. Disociar el tejido neural <ol><li> Pesar el tejido cerebral en un tubo que contiene 1 ml HBSS (w / o) </li><li> Transferencia de cerebro de ratón en el tubo C que contiene 1950 l de mezcla de enzimas precalentado 1. (NOTA: este protocolo está destinado a ≤ 400 mg, pero los volúmenes de solución se pueden ampliar para dar cabida a hasta 1600 mg de tejido cerebral por tubo C) </li><li> Coloque el tubo de C en el disociador gentleMACS y ejecutar el programa &quot;m_brain_01&quot;. </li><li> Incubar con la rotación (4 rpm con un rotor de tubo MACSmix) durante 15 minutos a 37 ° C. </li><li> Coloque el tubo de C en el disociador gentleMACS y ejecutar el programa &quot;m_brain_02&quot;. </li><li> Durante el paso de la disociación preparar 30 l de mezcla de enzimas del 2 por 400 mg de tejido con cuidado mezcla de 20 l de solución de 3 y 10 l de solución 4. </li><li> Añadir la mezcla de enzimas 2 del metro a través de la C-tabique sellado tapa y mezcle suavemente sin agitación. </li><li> Incubar el tubo C con rotación de 10 min a 37 ° C en una incubadora. </li><li> Coloque el tubo de C en el disociador gentleMACS y ejecutar el programa &quot;m_brain_03&quot;. </li><li> Incubar el tubo C con rotación de 10 min a 37 ° C en una incubadora. </li><li> Centrifugar brevemente para recoger la muestra en la parte inferior del tubo. </li></ol> 3. Filtración <ol><li> Seleccione un filtro lo suficientemente grandes para permitir el paso de las células de interés. Por ejemplo, las células de Purkinje son demasiado grandes para pasar a través de un filtro de 30 micras, pero Prominin-1 + células. </li><li> Use una pipeta de l suitable1000 para eliminar las células forma el tubo de C a través del tabique sellado la tapa y aplicarlo a la Pre-filtro de separación en un tubo de recogida de 15 ml. (NOTA: para mayor tamaño de la muestra utiliza un tubo de recogida de 50 ml). </li><li> Lavar el filtro con 10 ml de HBSS (w). </li><li> Centrifugar las células filtrada a 300 xg durante 10 min a temperatura ambiente. </li><li> Resuspender el sobrenadante en el medio o de amortiguamiento de la elección para aplicaciones posteriores. </li><li> Para eliminar los restos de mielina, la mielina utilizar bolas de eliminación. </li></ol> 4. Resultados representante: Por favor, véanse las Figuras 1-3 <img src="/files/ftp_upload/1267/Fig_1.png" alt="fig 1" /> Figura. 1 La disociación del cerebro con el disociador gentleMACS resultó en 97% las células viables, como lo muestra el flujo de análisis de citometría. <img src="/files/ftp_upload/1267/1267fig2.png" alt="fig 2" /> Figura. 2 imagen microscopio de luz de la formación de neuroesfera después de la clasificación de células magnético utilizando Anti-Prominin-1 MicroBeads después de 7 días de cultivo en MACS ® NeuroMedium complementado con MACS Suplemento B27 PLUS. Las células fueron preparados a partir de cerebro de ratón CD1 (P3), utilizando el tejido neural disociación Kit (P). <img src="/files/ftp_upload/1267/1267fig4.jpg" alt="fig 3.5" /> Figura. 3 restos de mielina en una sola célula suspensiones constituyendo una grave amenaza de aislamiento de células y la eliminación de los residuos de mielina por bolas eliminación de mielina aumenta la eficiencia de separación celular. Las separaciones de MACS con Anti-Prominin-1 MicroBeads, P22 cerebro de ratón se disocian con el tejido neural disociación Kit (P). Las células de la suspensión de una sola célula eran utilizados directamente para la separación, o se presentaron a la mielina agotamiento utilizando bolas de mielina eliminación. Comparación de la separación de las muestras con y sin la eliminación de mielina, demuestra que la pureza es mayor para las muestras con la eliminación de mielina anterior.

<ol>
	<li>Hardt, O., Scholz, C., K&#252;sters, D., Yanagawa, Y., Pennartz, S., Cremer, H., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+defines+differences+between+region-specific+GABAergic+neurons.">Gene expression analysis defines differences between region-specific GABAergic neurons.</a> Mol Cell Neurosci. 39, 418-428 (2008).</li><li>Lee, J. K., McCoy, M. K., Harms, A. S., Ruhn, K. A., Gold, S. J., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+of+G-protein+signaling+10+promotes+dopaminergic+neuron+survival+via+regulation+of+the+microglial+inflammatory+response.">Regulator of G-protein signaling 10 promotes dopaminergic neuron survival via regulation of the microglial inflammatory response.</a> J Neurosci. 28, 8517-8528 (2008).</li><li>K&#246;rnyei, Z., G&#243;cza, E., R&#252;hl, R., Orsolits, B., V&#246;r&#246;s, E., Szab&#243;, B., V&#225;govits, B., Madar&#225;sz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglia-derived+retinoic+acid+is+a+key+factor+in+glia-induced+neurogenesis.">Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis.</a> FASEB J. 21, 2496-2509 (2007).</li><li>Meylan, F., Davidson, T. S., Kahle, E., Kinder, M., Acharya, K., Jankovic, D., Bundoc, V., Hodges, M., Shevach, E. M., Keane-Myers, A., Wang, E. C., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TNF-family+receptor+DR3+is+essential+for+diverse+T+cell-mediated+inflammatory+diseases.">The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases.</a> Immunity. 29, 79-89 (2008).</li><li>Skog, J., W&#252;rdinger, T., van Rijn, S., Meijer, D. H., Gainche, L., Sena-Esteves, M., Curry, W. T., Carter, B. S., Krichevsky, A. M., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+microvesicles+transport+RNA+and+proteins+that+promote+tumour+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.</a> Nat Cell Biol. 10, 1470-1476 (2008).</li></ol>

Los autores son empleados de Miltenyi Biotec GmbH, Alemania.

El disociador gentleMACS facilita la preparación estandarizada de una sola célula suspensiones de tejidos nerviosos en un sistema cerrado. Neuronal del tejido Kits disociación están optimizados para conservar epítopos antígeno necesario para otras aplicaciones como immunostainings y separación celular inmunomagnética 1-5. En este protocolo se muestra la disociación enzimática suave del cerebro del ratón con el disociador gentleMACS y el tejido neural disociación Kit (P), produciendo en las células de un 97% viable. 100 mg de tejido nervioso entre los rendimientos de 5x10 6 y 1x10 7 células, dependiendo de la edad de los tejidos del huésped. El objetivo 1 Prominin células + fueron aislados usando Anti-Prominin-1 MicroBeads y luego fueron a la cultura. El protocolo se demuestra que es aún más eficaz con el uso adicional de bolas de eliminación de la mielina cuando se trabaja con el tejido neural derivados de ratones P7&gt;.

<table><tbody><tr><td>C Tubes</td><td>Miltenyi Biotec</td><td>Order no. 130-093-237</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&lt;/a&gt;</td></tr><tr><td>gentleMACS&amp;#8482; Starting Kit</td><td>Miltenyi Biotec</td><td>Order no. 130-093-235</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&lt;/a&gt;</td></tr><tr><td>MACSmix&amp;#8482; Tube Rotator</td><td>Miltenyi Biotec</td><td>Order no. 130-090-753</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&lt;/a&gt;</td></tr><tr><td>Neural Tissue Dissociation Kit (P)</td><td>Miltenyi Biotec</td><td>Order no. 130-092-628</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&lt;/a&gt;</td></tr><tr><td>Pre-Separation Filters</td><td>Miltenyi Biotec</td><td>Order no. 130-041-407</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx&lt;/a&gt;</td></tr><tr><td>Anti-Prominin-1 MicroBeads</td><td>Miltenyi Biotec</td><td>Order no. 130-092-333</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx&lt;/a&gt;</td></tr><tr><td>Anti-Prominin-1-APC</td><td>Miltenyi Biotec</td><td>Order no. 130-092-335</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx&lt;/a&gt;</td></tr><tr><td>Myelin Removal Beads</td><td>Miltenyi Biotec</td><td>Order no. 130-094-544</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx&lt;/a&gt;</td></tr><tr><td>Art 1000 Reach Pipet Tips </td><td>Molecular BioProducts</td><td>Order no. 2079</td><td>&lt;a href=&quot;http://www.mbpinc.com/ASP/products.aspx?query_TipID=61&quot;&gt;http://www.mbpinc.com/ASP/products.aspx?query_TipID=61&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.mbpinc.com/html/products/art/display.html&quot;&gt;http://www.mbpinc.com/html/products/art/display.html&lt;/a&gt;</td></tr><tr><td>Neural Tissue Dissociation Kit (T)</td><td>Miltenyi Biotec</td><td>Order no. 130-093-231</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx&lt;/a&gt;</td></tr><tr><td>Propidium Iodide Solution</td><td>Miltenyi Biotec</td><td>Order no. 130-093-233</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&lt;/a&gt;</td></tr></tbody></table>

generation of single-cell suspensions from mouse neural tissue

Dentro del sistema nervioso, cientos de tipos de células neuronales y gliales han sido descritos. Cada tipo específico de célula en el cerebro o la médula espinal tiene un repertorio de moléculas de superficie celular, o determinantes moleculares, a través del cual pueden ser identificados y caracterizados. En la actualidad, la identificación de células robusto y tecnologías de separación requiere una sola célula preparaciones que se generen al mismo tiempo limitar la muerte celular y la destrucción de la proteína de la superficie característica. El disociador gentleMACS, cuando se utiliza en combinación con tripsina o papaína más los paquetes de la disociación, puede con eficacia y suavidad disociar el tejido cerebral, mientras que la preservación de los epítopos del antígeno y la limitación de la pérdida de células. Preparación estandarizada de una sola célula suspensiones se consigue mediante tubos de C y los programas optimizados preestablecidos gentleMACS. Una vez generados, una sola célula suspensiones pueden ser tratadas con los conjugados monoclonales como el anti-Prominin-1 MicroBeads, que identifican progenitores neurales, o purificado utilizando bolas de mielina eliminación.

Watch this Scientific Journal Video about Generation of Single-Cell Suspensions from Mouse Neural Tissue at JoVE.com

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Disociar las células de tipos específicos de tejido requiere de parámetros específicos para aggitation tejido para obtener un alto volumen de células viables, cultivables. El Miltenyi gentleMACS disociador optimiza esta tarea con un protocolo simple y práctico. En esta publicación, el uso de este aparato en el tejido nerual se explica.

Generación de un solo suspensiones de células de tejido de ratón Neural

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

generation-of-single-cell-suspensions-from-mouse-neural-tissue

Research

JoVE Journal

Biology

22.4K vistas.  Miltenyi Biotec,GmbH.  中文 日本語 

Video: Generation of Single-Cell Suspensions from Mouse Neural Tissue

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Cultura de ratón con células madre neurales Precursores

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Investigación

Biología

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

El establecimiento de embriones de ratón con células madre neurales Cultura mediante el ensayo de neuroesfera

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neurociencias

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Generación de las células troncales nerviosas de desechados Humanos tejido cortical fetal

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

Un ratón, dos culturas: Aislamiento y cultivo de adultos Neural las células madre de las dos zonas neurogénicos de ratones individuales

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Aislamiento y cultivo de células madre adultas neural de la Zona subcallosal Ratón

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Aislamiento y cultivo de las células madre neurales embrionarias de ratón

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

<meta charset="utf8"></head><body>Nucleofección eficiente de NSCs aisladas de la zona subventricular murina

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>Autor destacado: Nucleofección mejorada para la entrega de genes de alta eficiencia en cultivos de células madre neurales derivadas de la zona subventricular murin

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Aislamiento, expansión y nucleofección de células madre neurales de la zona subventricular murina adulta

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

Obtención de suspensiones unicelulares de la médula ósea del cerebro y el cráneo murinos

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Autor destacado: Investigación de la compleja respuesta inmunitaria tras la isquemia cerebral

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

Aislamiento de células inmunitarias del cerebro y el cráneo de ratones

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...

Generación de un solo suspensiones de células de tejido de ratón Neural

Transcribir

Resumen

Explorar más videos

Capítulos en este video

Cultura de ratón con células madre neurales Precursores

El establecimiento de embriones de ratón con células madre neurales Cultura mediante el ensayo de neuroesfera

Generación de las células troncales nerviosas de desechados Humanos tejido cortical fetal

Un ratón, dos culturas: Aislamiento y cultivo de adultos Neural las células madre de las dos zonas neurogénicos de ratones individuales

Aislamiento y cultivo de células madre adultas neural de la Zona subcallosal Ratón

Aislamiento y cultivo de las células madre neurales embrionarias de ratón

Aislamiento, expansión y nucleofección de células madre neurales de la zona subventricular murina adulta

Aislamiento, expansión y nucleofección de células madre neurales de la zona subventricular murina adulta

Aislamiento, expansión y nucleofección de células madre neurales de la zona subventricular murina adulta

Aislamiento de células inmunitarias del cerebro y el cráneo de ratones