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

24.2K 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

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 ...

科研

神经科学

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 ...

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 ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

纤维蛋白富集和 tPA 敏感的光血栓形成卒中模型

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent ...

使用流式细胞术进行小鼠脑组蛋白修饰定量

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

作者聚焦:基因表达调控研究的增强

This work describes a protocol for the quantification of global histone modifications using intranuclear flow cytometry in isolated brain microglia. The work also contains the microglia isolation protocol that was used for data collection.

使用核内流式细胞术定量分离小鼠脑小胶质细胞中的整体组蛋白翻译后修饰

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to ...

从成年小鼠脊髓中高效提取星形胶质细胞和小胶质细胞

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

作者聚焦:从成年小鼠脊髓中分离神经胶质细胞 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...