这项工作是由退伍军人管理局优异审查补助金（NEUD-004-07F）（以SP）的支持。

1。扩展人类的NPC（ 图1） <ol><li>重振胎儿脑（龙沙，Walkersville，MD，USA），并将它们悬浮培养中的T-75瓶中含有增殖补充，表皮生长因子（10毫微克/毫升）的神经球（ 图1A）的Neurobasal培养基人类的NPC的冷冻股票和FGF（10纳克/毫升）。 </li><li>后在培养3天后，将神经球转移到15毫升管中，离心分离机，在500 rpm离心5分钟。 </li><li>弃去上清液留下〜100μl的培养基上的细胞沉淀，并转移到Eppendorf管中。将100μl细胞沉淀，就足以分裂成2个T-75瓶。如果少，减少瓶中的数量作为neuroprogenitor细胞不能增殖，如果分割薄。 </li><li>磨碎的沉淀用200μl的尖端50倍，同时保持对管底部的200μl的小费。分裂细胞悬液平均分成两部分，并将它们添加到2个T-75瓶（1-2分），一个用于继续扩张，另一个用于分化。 </li><li>通过改变培养基每4-5天，直到神经球达到300-500微米原始大小继续neuroprogenitor细胞扩张（第一瓶）的培养。通过离心改变介质（500转，5分钟），弃去旧培养基，并添加新的媒介。 </li></ol> 2。分化人类的NPC成神经元丰富的文化（ 图2） <ol><li>为分化neuroprogenitor细胞向神经元丰富的文化，将单盖玻片到24孔板中并涂覆有聚-L-赖氨酸的100微克/毫升的各孔中加入500微升每孔。孵育菜在室温30分钟。 </li><li>吸出聚-L-赖氨酸溶液中，用无菌水漂洗该板。 </li><li>接着，涂层的钢板用500μl的5μg/ ml的小鼠层粘连蛋白的孔中并孵育30分钟。孵育后，洗井用PBS。 </li><li> splitti后4天毫微小区（步骤1.4），转移将烧瓶中的小神经球标记为"对于分化&#39;到包被的培养皿以每孔〜500的神经球在24孔平板的密度。 </li><li> 6小时后，当神经球附着到培养皿，取出增殖培养基中，并添加选自Neurobasal培养基的分化培养基，B27添加剂，NGF（20纳克/毫升），BDNF（10毫微克/毫升）中，DBC（100毫米），和视黄酸（2μM）。 </li></ol> 3。差异化的NPC的免疫荧光染色（ 图3） <ol><li>以下neuroprogenitor细胞在神经分化培养基中两周的培养时，获得一个神经元的富培养。冲洗的神经元一旦在PBS中，然后修复它们在4％多聚甲醛30分钟。一旦固定，冲洗细胞三次，用PBS。 </li><li>孵育的细胞用透化缓冲液（5％BSA和0.2％的Triton X-100的PBS中）在室温下60分钟。 </li><li>孵育菜O / N在4°C I呐振动筛与多克隆和单克隆抗体的PBS中3％BSA的下列组合:突触素（1:250）的NeuN（1:250）和;乙酰胆碱酯酶（1:500）和突触素（1:250），BDNF（1 :500）和GAP43（1:250），STAT3（1:500）和GFAP（1:1,000）。 </li><li>用PBS洗涤盖玻片3次，然后孵育它们与抗兔Cy3和抗小鼠FITC二抗在室温下在黑暗中90分钟。孵育后，用PBS洗涤盖玻片3次。 </li><li>将10微升安装介质的载玻片上，取出盖玻片从培养皿用镊子，并把它倒挂在安装介质上。轻轻擦掉多余的安装介质和密封的边缘与指甲油。 </li><li>检查荧光显微镜的免疫染色的神经元。 </li></ol> 4。神经元的激光捕获显微切割（ 图4） <ol><li>分化的NPC成神经元丰富的文化对PEN膜幻灯片š以下对于图2中描述的方法用聚-L-赖氨酸和小鼠层粘连蛋白。 </li><li>执行无RNA酶的条件下，所有后续步骤。 </li><li>使用HistoGene染色（大角星），以可视化的细胞染色的文化。 </li><li>发现纯神经元群体缺乏使用与Veritas LCM系统的整个幻灯片的路线图图像星形胶质细胞等领域。使用绘图工具标记的神经元。 </li><li>执行使用计算机控制的精确度和自动化在一个两步骤过程中，这些区域的激光捕获。首先，IR（红外线）被射向多点为70毫瓦和2500微秒的脉冲激光功率设置，以获得膜附着到帽。接着，标记区域使用的UV刀具以10毫伏的低级别设置切除。 </li><li>这些工艺的组合选择性地捕获从PEN膜标记区域到CAPSURE LCM宏观帽。当盖子被提起，与n的膜eurons附着到帽。 </li><li>提取总RNA的使用PicoPure RNA提取试剂盒（大角星），LCM样品和治疗用DNase。 </li><li>使用RiboAMP RNA扩增试剂盒，按照从试剂盒说明书扩增分离出的RNA。 </li><li>利用TaqMan探针来检测人神经丝重链（华南金控）进行实时定量RT-PCR分析。 </li></ol>缩写: AD，阿尔茨海默氏病; BDNF，脑源性神经营养因子; CREB，环AMP应答元件结合蛋白; DBC，双丁酰环腺苷酸; EGF，表皮生长因子; FGF，成纤维细胞生长因子; GFAP，神经胶质纤维酸性蛋白; LCM，激光捕获显微切割，神经生长因子，神经生长因子，全国人大代表，neuroprogenitor细胞，PEN，聚萘二甲酸。

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没有利益冲突的声明。

我们在本研究中通过自我更新的人neuroprogenitor细胞分化神经元丰富的细胞培养模型和方法，通过激光捕获显微切割分离神经元的纯人口。我们已经使用了NGF，BDNF，DBC和视黄酸对的NPC的神经元分化的组合。 DBC是用来激活CREB，增强神经发生12的转录因子。维甲酸诱导细胞周期退出和减少胶质人口13。在这项研究中所描述的方法采用修改过我们之前的报告14。我们早先用捣碎的?...

寿命长的神经发生是已知发生在侧脑室的脑室下区和在成年哺乳动物脑1的齿状回的颗粒下层。 Neuroprogenitor细胞（的NPC）来自这些地区的起源是多能细胞，它可以分化成神经元，星形胶质细胞和少突胶质细胞2。的NPC所产生的，因为其潜在的患者的各种神经变性疾病，包括帕金森氏病，肌萎缩性侧索硬化，中风和阿尔茨海默氏病（AD）的3到被移植的兴趣。与研究的NPC一般都集中在这个移植的角度，但NPC来源的神经元的细胞培养模型，以确定神经退行性病变的机制的潜力还没有得到充分发挥。以前的研究一般用有丝分裂后的神经元从啮齿动物脑组织中需要被分离为每个实验中分离，因为它们是不自我更新。虽然人类成神经细胞瘤细胞系包括SH-SY5Y和SK-N-MC细胞可以扩展，它们不具有原代神经元的特征。人类的NPC，在另一方面，提供了两者的优点，因为它们可以被扩展为多通道，并且可以分化产生的细胞群体与原代神经元4,5的特性。在目前的研究中，我们描述了一种新的分化方案，从人胎脑分离市售的NPC获得一致的神经元的富人口。因为这些文化都包含一个小的百分比胶质细胞，我们需要更多的方法来分离神经元的分子鉴定纯人口。激光捕获显微切割（LCM）是一种新颖的技术，其中来自组织切片的细胞同源性的群体可以被选择性地捕获用于基因表达分析6。大脑是一个异类组织包括神经元，神经胶质细胞和其他细胞TYPES。 LCM被用于确定神经元特异性基因表达分析7-10。之前我们已经进行海马神经元的LCM从公元（Tg2576转）鼠脑切片，专门用于显示在海马神经元11表达降低的环磷酸腺苷反应元件结合蛋白（CREB）和BDNF。在本研究中，我们描述了人类的NPC的扩大，神经元分化，免疫荧光染色神经元标志物及LCM神经元对PEN膜玻片培养的分离程序。

<table><tbody><tr><td>Neuroprogenitor cells (NPCs) </td><td>Lonza</td><td>PT-2599</td><td></td></tr><tr><td>Neurocult NS-A human basal medium</td><td>Stem cell technology</td><td>5750</td><td></td></tr><tr><td>Neurocult NS-A Proliferation supplement</td><td>Stem cell technology</td><td>5753</td><td></td></tr><tr><td>Neurocult NS-A Differentiation supplement</td><td>Stem cell technology</td><td>5754</td><td></td></tr><tr><td>B-27 Supplement (50x) </td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>Human epidermal growth factor</td><td>Stem cell technology</td><td>2633</td><td></td></tr><tr><td>Fibroblast growth factor </td><td>Sigma</td><td>F0291</td><td></td></tr><tr><td>Brain Derived Neurotrophic Factor </td><td>Cell signaling</td><td>3897S</td><td></td></tr><tr><td>Nerve Growth Factor </td><td>Invitrogen</td><td>13257-019</td><td></td></tr><tr><td>Dibutyryl cyclic AMP</td><td>Sigma</td><td>D-0627</td><td></td></tr><tr><td>poly-L-lysine </td><td>Sigma</td><td>P-5899</td><td></td></tr><tr><td>Mouse laminin </td><td>Sigma</td><td>L-2020</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma</td><td>R-2625</td><td></td></tr><tr><td>STAT3 antibody</td><td>Cell signaling</td><td>9132</td><td></td></tr><tr><td>GFAP antibody</td><td>Cell signaling</td><td>3670</td><td></td></tr><tr><td>GAP43 antibody</td><td>Transduction laboratories</td><td>612262</td><td></td></tr><tr><td>BDNF antibody</td><td>Millipore</td><td>AB1534SP</td><td></td></tr><tr><td>Acetyl cholinesterase antibody </td><td>Santz cruz</td><td>Sc-11409</td><td></td></tr><tr><td>Synaptophysin antibody</td><td>Abcam</td><td>ab18008-50</td><td></td></tr><tr><td>NeuN antibody</td><td>Chemicon</td><td>MAB377</td><td></td></tr><tr><td>Synapsin antibody</td><td>Novus</td><td>NB300-104</td><td></td></tr><tr><td>Anti mouse FITC</td><td>Jackson Immuno</td><td>115-095-146</td><td></td></tr><tr><td></td><td>Research Laboratories</td><td></td><td></td></tr><tr><td>Anti Rabbit Cy3</td><td>Jackson Immuno</td><td>711-165-152</td><td></td></tr><tr><td></td><td>Research Laboratories</td><td></td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A1653</td><td></td></tr><tr><td>Triton-X 100</td><td>Acros</td><td>21568-2500</td><td></td></tr><tr><td>Paraformaldehye </td><td>Fisher</td><td>4042</td><td></td></tr><tr><td>Coverslip (Big circle cover slip)</td><td>Fisherbrand</td><td>12-545-102</td><td></td></tr><tr><td>Mounting medium (Prolong Gold)</td><td>Invitrogen</td><td>P36930</td><td></td></tr><tr><td>Pen membrane</td><td>Applied biosystems</td><td>LCM0521</td><td></td></tr><tr><td>Histogene LCM frozen section staining kit</td><td>Applied biosystems</td><td>KIT0401</td><td></td></tr><tr><td>RiboAmp RNA Amplification kit</td><td>Applied biosystems</td><td>KIT0201</td><td></td></tr><tr><td>Picopure RNA Isolation kit</td><td>Applied biosystems</td><td>KIT0202</td><td></td></tr><tr><td>CapsureMacro LCM caps</td><td>Applied biosystems</td><td>LCM0211</td><td></td></tr></tbody></table>

laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture

Neuroprogenitor细胞（的NPC）从人胎脑分离出的表皮生长因子（EGF）和成纤维细胞生长因子（FGF），以提供细胞的大量供应的存在下增殖的条件下展开。的NPC分化中的神经生长因子（NGF）的新组合的存在下，脑源性神经营养因子（BDNF），丁酰腺苷（DBC）与上餐盘涂有聚-L-赖氨酸和小鼠层粘连蛋白 - 视黄酸，以获得神经元丰富的文化。的NPC也有区别在缺乏神经营养因子，DBC和视黄酸和睫状神经营养因子（CNTF），得到星形胶质细胞培养丰富的存在。差异化的NPC的特点是免疫荧光染色的神经元标志物，包括的NeuN，突触素，乙酰胆碱酯酶，突触素及GAP43面板。胶质纤维酸性蛋白（GFAP）和STAT3，星形胶质细胞标志物，在分化的NPC的10-15％进行检测。为了便于细胞类型特异性分子鉴定，激光捕获显微切割进行分离神经元的聚乙烯酯（PEN）膜的载玻片培养。在这项研究中所描述的方法提供了有价值的工具，以促进我们的神经变性的分子机制的理解。

扩展的NPC（ 图1） 当神经球被分解为单细胞悬浮液通过研磨，枪头需要触摸在管的底部，以便有当将悬浮液用移液管上下一些阻力。中研磨的次数将个体，需要通过反复试验通过在显微镜下检查所得到的细胞悬液决定之间变化。关键是要提供细胞悬液足够的密度，因为扩散会以更快的速度，当的NPC都在密切接触。如果的NPC不生长，细胞密度应增加减少瓶中的数量。?...

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Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

人类neuroprogenitor细胞（的NPC）增殖的条件下扩大。的NPC分化成神经营养因子中的组合的存在下神经元的富培养物。用免疫组化法检测神经元标志物。为了分离神经元的纯人口的NPC分化的PEN膜幻灯片并进行激光捕获显微切割。

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

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Neuroscience

12.1K 次观看.  Denver VA Medical Center. 人类neuroprogenitor细胞（的NPC）增殖的条件下扩大。的NPC分化成神经营养因子中的组合的存在下神经元的富培养物。用免疫组化法检测神经元标志物。为了分离神经元的纯人口的NPC分化的PEN膜幻灯片并进行激光捕获显微切割。

视频: Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

发育生物学

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 ...

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

使用激光光镊操纵隔离神经细胞在体外

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

生物学

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

从开发小脑显微切割分离的不同的细胞群

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

预组装塑料微流体芯片中人类干细胞衍生神经元的分段化

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

分化后再蛋白的人类多能干细胞衍生神经元，用于高含量的内尿成生和突合性成熟筛查

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons

Source: Su, C. T. E., et al. <a href="https://app.jove.com/v/52408">An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System.</a> J. Vis. Exp. (2015).
This video demonstrates a method to differentiate human neural progenitor cells (NPCs) into neurons. The NPCs are introduced onto an established mouse astrocyte-rat cortical neuron co-culture, promoting NPC differentiation into neurons. The differentiation is confirmed by visualizing the cells under a confocal microscope.

一种将人神经祖细胞分化为神经元的共培养技术

1. Generation of Induced Pluripotent Stem Cells

	Culture human fibroblasts in DMEM supplemented with 10% fetal bovine serum (FBS) in 6-well plates.
	...