我们做出的贡献的雷纳塔佐野在构思的初步协议。 Ad&#39;A。主席赋予遗传学和基因治疗的儿童（JFC）持有的珠宝商。这部分工作是由美国国立卫生研究院拨款GM60905，DK52025及CA021764，和美国的黎巴嫩叙利亚相关慈善（ALSAC）。

<html>该协议的目的是MAMS和宝石鼠脑的分离和纯化解决方案需要分离线粒体，MAMS和宝石的分馏，得到粗线粒体准备: 溶液A:0.32 M蔗糖，1毫摩尔的NaHCO 3，1毫摩尔的MgCl 2，0.5mM的CaCl 2的 +蛋白酶抑制剂（添加新鲜的，根据需要） 溶液B:0.32 M蔗糖，1毫摩尔的NaHCO 3 +蛋白酶抑制剂（添加新鲜的，根据需要） MAMS隔离: 隔离媒介:250mM的甘露糖醇，5mM的HEPES pH为7.4，为0.5mM EGTA，0.1％BSA的 梯度缓冲液:225 mM的甘露糖醇，pH为7.5的25mM HEPES，1mM的EGTA，0.1％BSA的（最终浓度） 宝石隔离: 内容"&gt; 创业板提取缓冲液:25毫米HEPES pH值7.5，0.15 M氯化钠，1％的Triton X-100 +蛋白酶抑制剂（添加新鲜，） 创业板增溶缓冲液:50 mM Tris-盐酸，pH值8.8，5 mM的EDTA，1％SDS +蛋白酶抑制剂（添加新鲜的，需要的话）。 1。第一步亚细胞分离:去除的核，Unlysed细胞和细胞碎片<ol><li> CO 2室安乐死鼠标。 </li><li>立即移除大脑，减少一半，将在2毫升管上的冰和权衡。 </li></ol> 注意:在整个过程中，保持大脑半独立。以下内容适用于一个单一的半脑的治疗。 <ol start="3"><li>将一半大脑在预先冷冻的2毫升玻璃DOUNCE组织研磨机含有1ml冷的溶液A与一个大的间隙杵15总笔画均质。 </li><li>转移到15 ml的Falcon管中，在冰面上，并稀释匀浆高达10体积w / v的， 例如 0.225克至2.25毫升。 </li><li>离心样品在1400×g离心10分钟，在4℃下</li><li>小心除去上清液，转移至30毫升圆底玻璃离心管中，在冰上。确保不扰乱颗粒。 </li><li>将沉淀重悬于相同的10倍体积的解决方案。与3-6笔划一个小间隙杵，1毫升的时间在相同的研磨机，均质。 </li><li>转移到一个新的15毫升的猎鹰管和离心710×g离心10分钟，在4°C。这会导致在颗粒的细胞核和细胞碎片 。 </li><li>小心除去上清液和游泳池与保存在步骤1.6中的上清液。 </li></ol> 2。第二步:原油线粒体隔离<ol><li>离心上清液在13,800 xg离心10分钟，在4℃下</li><li>将上清转移到一个超清晰的贝克曼离心管中，在冰</li><li>沉淀重悬在10体积解。与3-6笔划一个小间隙杵，1毫升的时间在相同的研磨机，均质。 </li><li>离心步骤2.1。 </li><li>重复步骤2.2-2.4。 </li><li>由此产生的沉淀是一个丰富的线粒体组分。池上清液（细胞质和ER），用封口膜覆盖，并保持在冰上。 </li><li>与6冲程一个小间隙杵重悬沉淀，在4.8毫升/克（g被称为原始的脑重量）的溶液B，在一个新的均化器。 </li><li>使用玻璃巴氏吸管，准备蔗糖密度梯度超清晰贝克曼离心管，后来加入如下的试管底部，仔细，确保没有气泡形成: Resupended颗粒（0.32 M蔗糖） 3毫升850 mM的蔗糖（在1mM的NaHCO 3） 3毫升1M的蔗糖（在1mM的NaHCO 3） 3毫升1.2M的蔗糖（在1mM的NaHCO3） </li><li>在4℃下82,500 xg下离心2小时的渐变的结束时将得到的分离会产生三个频带和颗粒:1）的髓鞘和其​​他的膜污染物（0.32 M - 0.85 M接口），2）ER，高尔基体，血浆膜（0.85 M - 1 M接口）; 3）的突触体（1 M - 1.2 M接口）; 4）粗线粒体（粒料），用于随后的纯化步骤</li></ol> 3。线粒体相关的ER膜分离，MAMS <ol><li>重悬新鲜分离的原油从一个脑线粒体一半，在2毫升分离培养基中新加入的蛋白酶抑制剂。 </li><li>准备30％的的Percoll梯度 ， 梯度缓冲液8毫升，每半脑，和在超清晰贝克曼离心管的地方。 </li></ol> * 注意:请比较集中的梯度缓冲（1.43倍），以实现正确的最终浓度在Diluting珀可至30％。 <ol start="3"><li>在准备的梯度层线粒体悬浮液缓慢，以免任何气泡。 </li><li>离心30分钟，4℃，95,000 xg离心</li><li><ol class="lroman"><li> FIRST用玻璃巴斯德吸管移除重馏分（低级频带），并转移到新鲜的圆底玻璃管，在冰上。 </li><li>删除第二，另一个玻璃巴斯德吸管轻馏分（上带），并转移到一个单独的新鲜圆底玻璃管，在冰上。 </li></ol></li><li>稀释两个馏分收集在步骤3.5，用10毫升分离培养基和离心机在6300×g离心10分钟，在4℃下</li><li>从步骤3.6中的重质馏分弃上清，将沉淀重悬于另外10毫升分离培养基，并再次离心，3.6的步骤。将得到的沉淀将纯的线粒体组分 。 </li><li>将上清转移来自光压裂TION获得步骤3.6超清晰贝克曼离心管中，在冰上。弃沉淀。 </li><li>加入足够的隔离中等至步骤3.8中得到的上清液，以填补在100,000 xg离心1小时，4℃下的管和离心将所得的沉淀将是的MAMS馏分 。小心地取出，弃上清。 </li></ol> 注意:到了这一步，你也可以在10万XG离心1小时，4°C保存在步骤2.6上清。颗粒将是ER的部分 ，取上清液胞浆部分 。 4。提取的鞘糖脂富集微区，宝石<ol><li>在500μl的1毫升提取缓冲液中的20分钟，在冰上裂解纯线粒体和/或MAM馏分。 </li><li>离心裂解物在15,300×g离心2分钟，在4℃下</li><li>收集上清活性剂（Triton X-100的可溶性材料）和再离心微丸2分钟以除去残留的可溶性物质。活性剂（Triton X-100的提取线粒体和/或Triton X-100提取MAMS）。 </li><li>增溶缓冲液溶解的颗粒。这种溶解的材料代表创业板部分，并且可用于进一步的分析。 </li></ol>

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的作者有没有任何利益冲突的声明。

细胞内的膜或细胞器和细胞的质膜之间的站点之间的接触代表动态信令平台的基本的细胞过程。他们的生理和病理条件下的功能和组成的准确表征需要可靠和可重复的纯化方法。 MAMS和相应的宝石从成年小鼠的大脑的分离和纯化了专门的优化，通过我们的实验室方法详见这里。该协议已经成功实现了识别指定这些微区的分子效应，和背后的凋亡通路下游的Ca 2 +失衡导致神经细胞死亡的一种...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td></td><td></td><td></td><td> 分馏 </td></tr><tr><td>蔗糖</td><td> Fisher Scientific则</td><td> S5-500 </td><td></td></tr><tr><td>碳酸氢钠</td><td> Sigma-Aldrich公司</td><td> S-5761 </td><td></td></tr><tr><td>二氯化磷，镁六水</td><td> Fisher Scientific则</td><td> BP214-500 </td><td></td></tr><tr><td>氯化钙，二水</td><td> Sigma-Aldrich公司</td><td> C-5080 </td><td></td></tr><tr><td></td><td></td><td></td><td> MAM </td></tr><tr><td> D-甘露糖醇</td><td> Sigma-Aldrich公司</td><td> M9546-250G </td><td></td></tr><tr><td> HEPES </td><td> Fisher Scientific则</td><td> BP310-500 </td><td></td></tr><tr><td> EGTA </td><td> Sigma-Aldrich公司</td><td> E4378-250G </td><td></td></tr><tr><td> BSA，分数V，热休克，冻干</td><td>罗氏公司</td><td> 03-116-964-001 </td><td></td></tr><tr><td>珀</td><td>通用电器</td><td> 17-0891-02 </td><td></td></tr><tr><td></td><td></td><td></td><td> 创业板 </td></tr><tr><td>的Triton X-100的</td><td> Sigma-Aldrich公司</td><td> T9284-500毫升</td><td></td></tr><tr><td>氯化钠</td><td> Fisher Scientific则</td><td> S271-3 </td><td></td></tr><tr><td>三基地</td><td>罗氏公司</td><td> 03-118-142-001 </td><td></td></tr><tr><td>盐酸</td><td> Fisher Scientific则</td><td> A144S-500 </td><td></td></tr&gt; <tr><td> EDTA </td><td> Fisher Scientific则</td><td> BP120-500 </td><td></td></tr><tr><td>十二烷基硫酸钠（SDS）的</td><td> Fisher Scientific则</td><td> BP166-500 </td><td></td></tr><tr><td></td><td></td><td></td><td> 常见 </td></tr><tr><td>蛋白酶抑制剂片，完整无EDTA- </td><td>罗氏公司</td><td> 11-873-580-001 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td> 2毫升杜丝所有的玻璃组织研磨器</td><td>金布尔大通</td><td> 885300-0002 </td><td></td></tr><tr><td> 15毫升PP离心管，BD猎鹰</td><td> BD </td><td> 352097 </td><td></td></tr><tr><td> 30毫升的底部玻璃离心管</td><td>金布尔大通</td><td> 45500-30 </td><td></td></tr><tr><td> 15毫升的底部玻璃离心华夫格管</td><td>金布尔大通</td><td> 45500-15 </td><td></td></tr><tr><td>的超速管，超清晰，薄壁，14x89毫米</td><td>贝克曼 - 库尔特</td><td> 344059 </td><td></td></tr><tr><td>封口膜</td><td> Cole-Parmer公司</td><td> PM996 </td><td></td></tr><tr><td>一次性硼硅玻璃巴斯德吸管，9" </td><td> Fisher Scientific则</td><td> 13-678-20C </td><td></td></tr></tbody></table>

mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain

细胞内细胞器是高度动态的结构具有不同的形状和组合物，该组合物进行特定小区内在的和外在的线索。他们的膜通常定义的接触点，成为中心的交流信号分子和膜元件1,2,3,4并列。间细胞器的膜微区之间形成的内质网（ER）和线粒体在开幕式的IP3敏感的Ca 2 +通道被称为线粒体相关联的的ER膜或MAMS 4,5,6。蛋白质/脂质组成，这些膜接触位点的生化特性已被广泛研究，特别是在关系到他们的作用，调节细胞内Ca 2 + 4,5,6。的ER作为主存储区中细胞内Ca 2 +，和在这容量调节下游无数细胞过程的Ca 2 +信号，包括UDING翻译后的蛋白质折叠和蛋白maturation7。线粒体中，在另一方面，保持Ca 2 +的稳态，通过缓冲胞质Ca 2 +浓度，从而防止细胞凋亡途径的起始下游的Ca 2 +的不平衡4,8。的MAMS的动态特性使得他们的理想地点解剖的基本的蜂窝机制，包括Ca 2 +信号和调节线粒体Ca 2 +浓度，脂质的合成和运输，能量代谢和细胞的存活4,9,10,11,12。已经描述了几种协议用 ​​于纯化这些微区的从肝组织和培养的细胞13,14。 考虑到先前公布的方法，我们已经适应了从成年小鼠的大脑中的线粒体和MAMS隔离的协议。在这个过程中，我们增加了一个额外的纯化步骤，即的Triton X100抽取的，其中Enables糖脂丰富的微区的部分（"创业板"）的MAMS的隔离。这些创业板制剂共享几个蛋白质组分与小窝和脂筏，来自质膜或其他细胞内的膜，以及，提出充当收集聚类受体蛋白质点和用于蛋白质-蛋白质相互作用4,15。

根据我们的经验，使用这个协议，我们可以放心地推荐它从小鼠脑的MAMS，宝石和线粒体组分的分离和纯化。所概述的过程是高度重复性和一致性。在图1中，我们显示的途纯的线粒体和MAMS层上的Percoll梯度（3.4）的代表图像。纯化阿义，含乳白色频带的线粒体（三刀-P），在超离心管的底部偏析，当MAM馏分拼成一个以上的线粒体的漫反射和宽频带。仔细回收个别梯度带后，再悬浮在纯?...

Watch this Scientific Journal Video about Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain at JoVE.com

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

此过程说明了如何分离从成年小鼠的大脑相关的线粒体的ER膜或MAMS和糖脂富集的微区部分从MAMS和线粒体的准备工作。

线粒体相关的ER膜（MAMS）和鞘糖脂丰富的微区（GEMS）:分离小鼠脑

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

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Neuroscience

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

24.3K 次观看.  St Jude Children's Research Hospital. 此过程说明了如何分离从成年小鼠的大脑相关的线粒体的ER膜或MAMS和糖脂富集的微区部分从MAMS和线粒体的准备工作。

视频: Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

科研

神经科学

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

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 of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

产后神经球的分离和扩张 （P1+3） 小鼠神经原性球

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

线粒体相关的ER膜（MAMS）和鞘糖脂丰富的微区（GEMS）:分离小鼠脑

摘要

探索更多视频

此视频中的章节

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

从胚胎前脑内皮细胞的分离和培养

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

分离和成人神经干细胞的培养从鼠标Subcallosal区

新鲜人脑组织中毛细血管的分离

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

产后神经球的分离和扩张（P1+3）小鼠神经原性球

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

线粒体相关的ER膜（MAMS）和鞘糖脂丰富的微区（GEMS）:分离小鼠脑

摘要

探索更多视频

此视频中的章节

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

从胚胎前脑内皮细胞的分离和培养

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

分离和成人神经干细胞的培养从鼠标Subcallosal区

新鲜人脑组织中毛细血管的分离

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

产后神经球的分离和扩张 （P1+3） 小鼠神经原性球

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

产后神经球的分离和扩张（P1+3）小鼠神经原性球