Name: generation of single-cell suspensions from mouse neural tissue
Uploaded: 2009-07-07T19:00:00.000+00:00
Duration: 11 min 17 s
Description: Watch this Scientific Journal Video about Generation of Single-Cell Suspensions from Mouse Neural Tissue at JoVE.com

Per lavorare in condizioni sterili, si raccomanda di eseguire tutti i passaggi in una cappa a flusso laminare. 1. Materiale <ul><li> Neurali del tessuto dissociazione Kit (P) (Componenti: Soluzione 1,2, 3 e 4, buffer di memoria) </li><li> Pre-filtri di separazione </li><li> (Opzionale) Mielina rimozione Beads </li><li> Beta-mercaptoetanolo </li><li> gentleMACS dissociatore </li><li> C Tubi </li><li> MACSmix tubo Rotator </li><li> HBSS (w / o) </li><li> HBSS (w) </li></ul><ol><li> Preparare le seguenti soluzioni prima di iniziare la procedura: <ol><li> Sale Hanks &#39;Soluzione tampone senza cationi bivalenti Ca 2 + o Mg 2 + (HBSS (w / o) </li><li> HBSS, standard, cioè con Ca 2 + e Mg 2 + (HBSS (w) </li></ol></li><li> A seconda del epitopo dell&#39;antigene di interesse per le applicazioni successive, utilizzare la papaina a base di tessuto neurale dissociazione Kit (P) o la tripsina a base di tessuto neurale dissociazione Kit (T). </li><li> Preparare le seguenti soluzioni dal tessuto neurale dissociazione Kit: <ol><li> Soluzione 2: aggiungere beta-mercaptoetanolo a 0,067 mM </li><li> Soluzione 4: sciogliere polvere in 0,7 ml di buffer di archiviazione (fornito nel kit), mescolare delicatamente (non vortex) </li><li> Enzyme Mix 1: Pipettare 1,9 mL Soluzione 2 e 50 ul di soluzione 1 (entrambi da kit) in un tubo C e incubare per 10-15 minuti a 37 ° C. Questo è sufficiente a dissociarsi 400 mg di tessuto cerebrale. </li></ol></li></ol> 2. Dissociare il tessuto nervoso <ol><li> Pesare tessuto cerebrale in una provetta contenente 1 ml HBSS (w / o) </li><li> Trasferimento cervello di topo nel tubo C contenente 1950 ml di Enzyme Mix preriscaldato 1. (NOTA: questo protocollo è scritto per ≤ 400 mg, ma volumi di soluzione può essere scalata a ospitare fino a 1600 mg di tessuto cerebrale per tubo C) </li><li> Posizionare il tubo C sul dissociatore gentleMACS ed eseguire il programma &quot;m_brain_01&quot;. </li><li> Incubare con rotazione (4 giri al minuto usando un tubo Rotator MACSmix) per 15 minuti a 37 ° C. </li><li> Posizionare il tubo C sul dissociatore gentleMACS ed eseguire il programma &quot;m_brain_02&quot;. </li><li> Durante la fase di dissociazione preparare 30 ml di Enzyme Mix 2 per 400 mg di tessuto con delicatezza mescolando 20 ml di soluzione 3 e 10 ml di soluzione 4. </li><li> Aggiungi Enzyme Mix 2 al tubo C attraverso il setto-sigillato tappo e mescolare delicatamente senza vortex. </li><li> Incubare la provetta C con rotazione per 10 minuti a 37 ° C in un incubatore. </li><li> Posizionare il tubo C sul dissociatore gentleMACS ed eseguire il programma &quot;m_brain_03&quot;. </li><li> Incubare la provetta C con rotazione per 10 minuti a 37 ° C in un incubatore. </li><li> Centrifugare brevemente per raccogliere il campione al fondo della provetta. </li></ol> 3. Filtrazione <ol><li> Selezionare un filtro abbastanza grande per consentire il passaggio alle cellule di interesse. Per esempio, cellule di Purkinje sono troppo grandi per passare attraverso un filtro di 30 micron, ma Prominin-1 + cellule. </li><li> Utilizzare una pipetta suitable1000 microlitri per rimuovere le cellule formano il tubo C il setto-tappo sigillato e applicarlo al pre-filtro di separazione su un tubo di raccolta da 15 ml. (NOTA: per le dimensioni del campione più ampio utilizzare un tubo di raccolta da 50 ml). </li><li> Lavare il filtro con 10 ml di HBSS (w). </li><li> Centrifugare le cellule filtrata a 300 xg per 10 minuti a temperatura ambiente. </li><li> Risospendere il supernatent nel medio o tampone di scelta per le applicazioni successive. </li><li> Per rimuovere i detriti della mielina, utilizzare Beads rimozione mielina. </li></ol> 4. Risultato rappresentante: Si prega di vedere figure 1-3 <img src="/files/ftp_upload/1267/Fig_1.png" alt="fig 1" /> Figura. 1 La dissociazione cervello con il dissociatore gentleMACS portato a 97% cellule vitali, come dimostra la citometria a flusso. <img src="/files/ftp_upload/1267/1267fig2.png" alt="fig 2" /> Figura. 2 foto microscopio Luce di formazione neurosfere dopo l&#39;ordinamento delle cellule magnetico utilizzando Anti-Prominin-1 microsfere dopo 7 giorni di coltivazione in MACS ® NeuroMedium integrato con MACS supplemento B27 PLUS. Le cellule sono state preparate dal CD1 cervello di topo (P3) con il tessuto neurale dissociazione Kit (P). <img src="/files/ftp_upload/1267/1267fig4.jpg" alt="Fig. 3.5" /> Figura. 3 detriti mielina in cella singola sospensioni un notevole pericolo per l&#39;isolamento delle cellule, e la rimozione di detriti mielina da Perline rimozione mielina separazioni aumenta l&#39;efficienza delle cellule. Per le separazioni MACS con Anti-Prominin-1 microsfere, P22 cervello di topo era dissociato con il tessuto neurale dissociazione Kit (P). Cellule dalla cella singola sospensione sono state direttamente utilizzate per la separazione, o sono stati sottoposti al depauperamento della mielina con perline di rimozione mielina. Confrontando la separazione da campioni, senza e con la rimozione della mielina, dimostra che la purezza è maggiore per i campioni con la rimozione della mielina precedente.

<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>

Gli autori sono dipendenti di Miltenyi Biotec GmbH, Germania.

Il dissociatore gentleMACS facilita la preparazione standardizzata di singole cellule sospensioni dai tessuti neurali in un sistema chiuso. Neurali del tessuto kit dissociazione sono ottimizzati per preservare epitopi antigene necessaria per ulteriori applicazioni, come immunostainings e la separazione delle cellule immunomagnetica 1-5. In questo protocollo si mostra la dissociazione dolce enzimatica di cervello di topo utilizzando il dissociatore gentleMACS e il tessuto neurale dissociazione Kit (P), ottenendo nel 97% cellule vitali. 100 mg di rendimenti tra il tessuto neurale 5x10 1x10 6 e 7 celle, a seconda dell&#39;età del tessuto ospite. Il mirati Prominin-1 + sono state isolate le cellule utilizzando Anti-Prominin-1 microsfere e successivamente presi in cultura. Il protocollo è dimostrato di essere ancora più efficace con l&#39;uso aggiuntivo di Perline rimozione mielina quando si lavora con i tessuti neurali derivate da topi 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

All&#39;interno del sistema nervoso, centinaia di tipi di cellule neuronali e gliali sono stati descritti. Ogni tipo di cellule specifiche del cervello o del midollo spinale ha un repertorio di molecole di superficie cellulare, o determinanti molecolari, attraverso i quali può essere identificato e caratterizzato. Attualmente l&#39;identificazione delle cellule robuste e richiedono tecnologie di separazione a cella singola preparati da generare al tempo stesso limitare la morte cellulare e la distruzione delle proteine ​​di superficie caratteristico. Il dissociatore gentleMACS, se usato in combinazione con tripsina o la papaina kit a base di dissociazione, può efficacemente e delicatamente dissociare tessuto cerebrale, preservando epitopi dell&#39;antigene e limitare la perdita di cellule. Preparazione standardizzata di singola cellula sospensioni è ottenuta utilizzando tubi C e ottimizzato, programmi preimpostati gentleMACS. Una volta generato, cella singola sospensioni possono essere trattati con coniugati monoclonali come Anti-Prominin-1 microsfere, che identificano progenitori neurali, o ulteriormente purificato utilizzando perline di rimozione mielina.

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

Dissociare cellule di tipi di tessuto specifico richiede parametri specifici per aggitation tessuto per ottenere un elevato volume di vitali, cellule coltivabili. Il Miltenyi gentleMACS dissociatore ottimizza questa operazione con un semplice protocollo pratico. In questa pubblicazione l&#39;utilizzo di questo apparecchio sul tessuto nerual è spiegato.

Generazione di Single-Cell Sospensioni dal tessuto neurale del mouse

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 ...

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JoVE Journal

Biology

22.4K Views.  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 di cellule precursori staminali neurali del mouse

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

Ricerca

Biologia

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.

Stabilire embrionali di topo Neural Stem Cell Culture del saggio di neurosfere

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 ...

Neuroscienze

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.

Generazione di cellule staminali neurali da scartate tessuto fetale umano corticale

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 mouse, due culture: isolamento e la coltura di Adult Neural Stem Cells dalle due zone neurogena Mice individuale

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.

Isolamento e la coltura di adulti neurali cellule staminali dal Subcallosal Zona mouse

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.

Isolamento e coltura di cellule staminali neurali embrionali del Mouse

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>Nucleofecazione efficiente di NSC isolate dalla zona subventricolare 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>Autore in primo piano: Nucleofection migliorata per la consegna genica ad alta efficienza in colture di cellule staminali neurali munine derivate dalla zona subven

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.

Isolamento, espansione e nucleofezione di cellule staminali neurali dalla zona subventricolare 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 ...

Ottenimento di sospensioni a cellula singola dal cervello murino e dal midollo osseo del cranio

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.

Autore in primo piano: Indagine sulla complessa risposta immunitaria a seguito di ischemia cerebrale

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.

Isolamento delle cellule immunitarie dal cervello e dal cranio del topo

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

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Isolamento delle cellule immunitarie dal cervello e dal cranio del topo