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

若要在无菌条件下，它是建议执行层流罩中的所有步骤。 1。材料<ul><li>神经组织解离套件（P）（组件：解决方案1,2，3，4，存储缓冲器） </li><li>预分离过滤器</li><li> （可选）髓鞘去除珠</li><li> β-巯基乙醇</li><li> gentleMACS Dissociator </li><li>彗星管</li><li> MACSmix管肩</li><li>的HBSS（W / O） </li><li>的HBSS（W） </li></ul><ol><li>准备开始前的协议以下解决方案： <ol><li>汉克斯“没有二价阳离子的缓冲盐溶液中的Ca 2 +或Mg 2 +（HBSS（W / O ） </li><li> HBSS中，标准的，即与Ca 2 +和Mg 2 +（HBSS（W） </li></ol></li><li>根据不同的抗原表位，在随后的应用程序的兴趣，使用的木瓜蛋白酶为基础的神经组织解离套件（P）或胰蛋白酶为基础的神经组织离解包（T）。 </li><li>从神经组织离解包准备以下解决方案： <ol><li>解决方案2：添加β-巯基乙醇，以0.067毫米</li><li>解决方案4：粉末溶于0.7毫升存储缓冲区（试剂盒提供），轻轻混匀（不要旋涡振荡） </li><li>酶混合物1：移液器1.9毫升溶液中2到C管和孵育10-15分钟50μL解决方案1（套件），在37 ° C。游离于400毫克的脑组织，这是足够的。 </li></ol></li></ol> 2。游离的神经组织<ol><li>称取脑组织中含有1毫升的HBSS（W / O）管</li><li>鼠脑转移到C包含1950年μL预热酶混合1的管。 （注：这个协议是书面为≤400毫克，但卷可以扩展的解决方案，以适应脑组织至1600毫克，C管） </li><li> C管放置到gentleMACS Dissociator和运行方案“m_brain_01”。 </li><li>旋转（4 RPM使用MACSmix管肩）为15分钟，在37 ° C。 </li><li> C管放置到gentleMACS Dissociator和运行方案“m_brain_02”。 </li><li>在解离步骤准备30酶混合，每400毫克组织2液，轻轻混匀20μL的解决方案3和10μL解决方案4。 </li><li>隔密封帽通过添加酶混合物2到C管和无震荡轻轻混匀。 </li><li>孵育37℃10分钟旋转管°孵化器中的C。 </li><li> C管放置到gentleMACS Dissociator和运行方案“m_brain_03”。 </li><li>孵育37℃10分钟旋转管°孵化器中的C。 </li><li>短暂离心收集管底部的样品。 </li></ol> 3。过滤<ol><li>选择一个过滤器大到足以让感兴趣的细胞通过。例如，浦肯野细胞是太大，通过一个30微米的过滤器，但Prominin - 1 +细胞。 </li><li>使用suitable1000μL移液器取出细胞，隔密封帽的形式，通过C管，并将其应用到15毫升收集管上的预分离过滤器。 （注：较大的样本量使用50毫升收集管）。 </li><li>的HBSS（W）10毫升清洗过滤器。 </li><li>离心机在室温下10分钟在300 XG细胞过滤。 </li><li>悬浮在您的介质或为随后的应用程序选择的缓冲区上清。 </li><li>要删除髓鞘碎片，使用髓鞘去除珠。 </li></ol> 4。代表性的结果：请参见图1-3 <img src="/files/ftp_upload/1267/Fig_1.png" alt="图1" /> 图。 1 gentleMACS Dissociator大脑解离，导致97％的活细胞，流式细胞仪分析显示。 <img src="/files/ftp_upload/1267/1267fig2.png" alt="图2" /> 图。 2光镜下神经球形成后7天种植MACS磁性细胞分选后的图片使用反Prominin - 1微珠® NeuroMedium MACS补充B27的PLUS的补充。 CD1小鼠大脑中的神经组织解离套件（P）（P3），准备从细胞。 <img src="/files/ftp_upload/1267/1267fig4.jpg" alt="图3.5" /> 图。 3髓鞘碎片单细胞悬液，大大损害细胞的分离，髓鞘去除珠去除髓鞘碎片，提高了效率，细胞分离。互委会使用反Prominin - 1微球的分离，P22鼠脑分离使用的神经组织解离套件（P）。从单细胞悬液，细胞分离，可以直接使用，或提交髓鞘枯竭使用髓鞘去除珠。比较与无髓鞘去除样品的分离，表明与以前的髓鞘去除样品纯度较高。

<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>

作者是德国美天旎生物技术公司，雇员。

gentleMACS Dissociator有利于从一个封闭的系统中的神经组织的单细胞悬浮液的标准化准备。神经组织解离套件进行了优化，保留进一步应用，如immunostainings和免疫细胞的分离1-5需要的抗原表位。在这个协议中，我们展示了鼠标使用gentleMACS Dissociator和神经组织解离套件（P）的大脑的温和酶解，收益率在97％活菌。 5X10 6和1 × 10 7细胞，取决于宿主组织岁之间的神经组织产生100毫克。使用反Prominin - 1微珠针对性Prominin - 1 +细胞进行分离和随后进入的文化。该协议是从小鼠&gt; P7源性神经组织工作时，要额外使用髓鞘去除珠更有效。

<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

在神经系统，数百个神经元和神经胶质细胞类型进行了描述。特定的细胞类型中的每个大脑或脊髓有一个细胞表面的分子或分子的决定因素，通过它可以识别和特点的剧目。目前，强大的细胞识别和分离技术的要求，同时限制了细胞的死亡和破坏特征的表面蛋白产生单细胞的准备工作。 gentleMACS Dissociator，胰蛋白酶或木瓜蛋白酶为基础的离解包的组合使用时，可以有效地，轻轻地分解脑组织，同时保留抗原表位，并限制细胞的损失。使用C管和优化，预设的gentleMACS程序实现标准化制备单细胞悬液。一旦产生，单细胞悬浮液是可以治疗的单克隆共轭反Prominin - 1微球，并确定神经祖细胞或纯化进一步使用髓鞘去除珠一样。

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

游离从特定的组织类型细胞需要组织aggitation特定的参数，以获得一个可行的，可培养细胞的高容量。美天旎gentleMACS Dissociator优化这个任务简单，实用的协议。在本出版物中使用本仪器nerual组织进行了解释。

从小鼠神经组织产生单细胞悬浮液

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

22.5K 次观看.  Miltenyi Biotec,GmbH.  中文 日本語 

视频: 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.

培养小鼠神经干细胞的前体

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

科研

生物学

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.

建立小鼠胚胎神经干细胞培养神经球含量

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 ...

神经科学

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.

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

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.

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

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.

分离和成人神经干细胞的培养从鼠标Subcallosal区

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.

胚胎小鼠神经干细胞的分离与培养

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>对从小鼠脑室下区分离的 NSC 进行高效成核

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>作者聚焦：改进的核转染技术，在小鼠脑室下区衍生的神经干细胞培养物中实现高效基因递送

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.

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

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 ...

从小鼠脑和颅骨骨髓中获得单细胞悬液

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.

作者聚焦：研究脑缺血后的复杂免疫反应

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.

从小鼠大脑和颅骨中分离免疫细胞

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

从小鼠神经组织产生单细胞悬浮液

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培养小鼠神经干细胞的前体

建立小鼠胚胎神经干细胞培养神经球含量

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

分离和成人神经干细胞的培养从鼠标Subcallosal区

胚胎小鼠神经干细胞的分离与培养

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

来自成体小鼠脑室下区的神经干细胞的分离、扩增和成核转染

从小鼠大脑和颅骨中分离免疫细胞