这项研究是从本科生研究机会计划在UCI（AR和SP）赠款和赠款，从加州阿尔茨海默氏病的倡议和国家卫生部授予机构没有国家的支持。 HD38466，阿尔茨海默氏症研究中心授予没有。 AG16573（JB）

1。皮层组织块冷冻保存 材料的制备 <ol><li>准备无菌水（1:9）的10倍原液稀释1X汉克的缓冲盐溶液（HBSS）。在摄氏4度商店的HBSS。 </li><li>准备10％DMSO冻存液稀释的HBSS（1:9）的二甲基亚砜。冻结介质应该是取得了新的冷冻保存前直到冷冻储存在摄氏4度。 </li><li>钢刀片是用于组织系统斩波。在使用之前，刀片被淹没在70％乙醇为2小时消毒。立即组织处理前，无菌水冲洗3次刀片。避免留在无菌水的剃须刀过多的时间，因为它很容易氧化。 </li><li>一个NALGENE冷冻集装箱装入离心管（2.5毫升），然后带进无菌生物柜。 1毫升的冰鲜冷冻介质添加到每一瓶。冷冻集装箱，然后放置在摄氏4度至少2小时。 </li></ol> 清洗，切菜，和冻结 <ol><li>被冻结的脑组织，脑膜膜和血管的清洁。使用无菌针尖小心地取下冰袋上工作时从组织的碎片。清理组织应轻轻冲洗与冷的HBSS，并转移到一个新的Petri砍菜。 </li><li>冰袋上工作，使用消毒的刀片，迅速 ​​组织切成约1毫米的3块。清理组织工作的一小部分，在更好地控制在切菜过程中的时间结果，产生更均匀的块大小。 </li><li>在50 mL锥形管，慢慢地加入到50毫升的HBSS切碎组织。轻轻冲洗培养皿与HBSS中，以收集任何剩余的组织块。允许组织块下降到试管底部，建立一个松散的组织块颗粒。 </li><li>虽然该组织是解决生物安全柜，把先前冷冻的冷冻集装箱和冷冻管。摘帽所有的离心管，以加速加入组织的过程中。 </li><li>所有的组织块纷纷落户后，轻轻吸出多余的HBSS的，留下一个非常薄的层以上的颗粒的介质。离心气馁，因为它会导致组织块要坚持对方，冻结效率显着减少。 </li><li>使用广泛窍（切割用无菌剪刀尖）的枪头，慢慢地从底部的松散组织块颗粒收集200微升。的材料转移到一个离心管，从底部的沉淀下，再次收集组织。重要的是整个组织分配过程不超过3-4分钟每冷冻集装箱。这将确保该组织是暴露只是一个时间的冻结前的短期内，以二甲基亚砜。组织转移到小瓶后，放置至少4小时，在A - 80℃冷冻的冷冻集装箱。另外，冷冻集装箱可以在一夜之间，在-80℃。重复这个过程中，任何剩余的冷冻集装箱。 </li><li>从冷冻集装箱的冷冻管（S）转移到一个cryobox和长期储存在液氮罐。 </li></ol> 2。解冻和冷冻皮层组织块的培养 材料的制备 <ol><li>一天前解冻的组织样本，聚- L -赖氨酸涂布培养皿/盖玻片准备。皮层神经元在一般情况下，我们准备用500微克/毫升或1毫克/毫升涂菜肴。加入足够的聚- L -赖氨酸溶液（硼酸盐缓冲液中），全面覆盖的菜底部。如果需要盖玻片，确保它们完全淹没下聚- L -赖氨酸的解决方案。孵育至少12小时。在使用之前，彻底的菜肴与自由主义的无菌水冲洗3次，每次5 min。 </li><li>暖的HBSS是用来清洁解冻组织以及组织分解成细胞悬液。取决于解冻组织的需要的HBSS数量会有所不同，但通常为1离心管会稀释在50毫升的HBSS，并将于10毫升的HBSS分离。 </li><li>贝科的改良Eagle中等10％的铁补充牛小牛血清（EBS）和1％的抗生素/ antimycotic（AA）的混合物（DMEM培养液(++))应放置在37℃水浴。 </li><li>电镀液（注(+++))应在使用前新鲜。这种介质是准备加入抗生素/ antimycotic加1％B27和N2补充剂。 </li></ol> 注:媒体名称与穿越（+）追加指示列入媒体的碱基组成的添加剂。在这个文本中，DMEM培养液（+）表示DMEM培养液加10％EBS的加1％，AA，而NB (+++)表示注:PL我们的1％AA加B27的加氮气。 解冻和培养 <ol><li>从液氮罐取出离心管，并迅速解冻在37 ° C直到水浴中只有一小冰粒是观察里面的小瓶内容。 </li><li>轻轻转移的内容，以温暖的HBSS 50毫升的小瓶，在一个锥形管。使用宽口尖轻轻驱逐任何剩余的组织块的离心管中卡住。反转管的3-4倍，并允许组织在底部慢慢收集。组织应该迅速解决，但如果块太小，这可能不会发生光离心（〜200 ×克）建议。 </li><li>吸干多余的HBSS。 </li><li>添加10毫升温暖的HBSS组织颗粒和300μL0.25％胰蛋白酶和50μLDNAase。在37℃水浴孵育5分钟，然后在轨道摇床设置为80 rpm和37 ° C的一个额外的5分钟。 </li><li>这一时期后，将进入生物安全柜的组织块，并用10 mL的吸管仔细搅动的组织块，直到形成阴天细胞悬液。停用温暖的DMEM培养液中添加10毫升的胰蛋白酶（+）的细胞悬液。 </li><li> 1200 × 克 5分钟离心分离细胞。吸弃上清，在10毫升的温暖的DMEM重悬细胞沉淀（+ +）和量化使用血球细胞数。 </li><li>聚- L -赖氨酸涂层菜肴板细胞。通常，一个60毫米的菜将被镀与1 × 10 6细胞。 </li><li>允许细胞组织培养箱培养约1小时的菜/盖玻片的重视。建议在一个板块监测的附件的进展，每10分钟，直到有效附件。然后，轻轻地更换新鲜的DMEM (++).中期</li><li> 24小时后，更换的DMEM（+）用温水注(+++).部分介质的变化（50％）进行每5天用新鲜的NB (+++).健康的文化通常24小时后，进程的分化和增长的迹象。文化在这些条件下，执行部分介质的变化，每4-5天，可维持长时间的时间可达4-6周。 </li><li>对于生成的神经前体细胞（筹备），类似的协议，离心后，没有EBS的DMEM培养液的异常。细胞是镀金的T - 25的DMEM/F12（1:3），B27与表皮生长因子（20毫微克/毫升）的补充，让neuropheres形成烧瓶。要区分的NPC，神经球镀金层粘连蛋白（10微克/毫升）的DMEM/F12（1:3）培养基用N2涂盖玻片。 </li></ol> 3。代表性的成果健康的文化会表现出显着的生长和分化，后5天，解冻后，通常会在其增长稳定10天（图1）。这些文化Immuncocytochemical染色揭示众多的星形胶质细胞（图2A）和神经元对人类和大鼠原代神经元的文化（图2B）。此协议也适合对NPC的一代自由浮动的神经球（图3A），区分条件下，高品质（图3B，C）的混合培养的结果。 <img src="/files/ftp_upload/2384/2384fig1.jpg" alt="图1" /> 图1。冷冻人脑皮层组织细胞培养 ，冷冻的人脑皮层组织块接种于多聚赖氨酸涂布盖玻片解冻，生长10天。白天5细胞扩张明显，达到10天汇合。比例尺:100微米。 <img src="/files/ftp_upload/2384/2384fig2.jpg" alt="图2" /> 图2。细胞培养免疫细胞化学染色（A）培养染色标记的胶质神经胶质纤维酸性蛋白（GFAP，厚箭头）。 （二）发现神经元与神经元的标记β-微管蛋白III（TIII，细箭头）。 （三）人类和鼠文化展示丰富的神经胶质细胞和神经元后的10天中文化。一抗:鼠抗GFAP（1:1000）和兔抗TIII（1:1000）。二抗:抗鼠Alexa的594（1:500）和抗兔Alexa的488（1:500）。核染色:Hoestch蓝色（1:1000）。比例尺:50微米。 <img src="/files/ftp_upload/2384/2384fig3.jpg" alt="图3" /> 图3。代神经球。（一）冰冻组织处理，如上所述，并允许传播神经球。 （二）神经球被镀上盖玻片与众不同的条件下生长10天，导致细胞从神经球迁移。 （三）这两种神经元和星形胶质细胞是目前在区分神经干扩大边缘。核counterstaining，如在图2中使用的小学和中学的抗体。

<ol>
	<li>Robbins, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+brain+tissue.">Cryopreservation of human brain tissue.</a> Exp Neurol. 107, 208-213 (1990).</li><li>Ware, C. B., Nelson, A. M., Blau, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled-rate+freezing+of+human+ES+cells.">Controlled-rate freezing of human ES cells.</a> Biotechniques. 38, 879-880 (2005).</li><li>Paynter, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practical+issues+for+cryopreservation+of+nerve+cells.">Principles and practical issues for cryopreservation of nerve cells.</a> Brain Res Bull. 75, 1-14 (2008).</li><li>Thirumala, S., Gimble, J. M., Devireddy, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+stromal+vascular+fraction+of+adipose+tissue+in+a+serum-free+freezing+medium.">Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.</a> J Tissue Eng Regen Med. 4, 224-232 (2010).</li></ol>

冷冻保存提供了一个宝贵的脑组织样本，以供将来使用银行的机会。在这里，我们描述了一个简单而有效的的协议，同时生成神经元丰富的文化和神经前体细胞从冷冻的脑组织块。这种经济的过程避免了传统的冷冻保存技术，利用更昂贵的速率控制冰柜的成本。该协议允许可行的解冻后的神经元文化的一代提供一个快速而有效的的手段，以冻结组织块。整个冻结过程中可以采取低至20分钟。此外，使用这种方法组织块的初级神经元文化也可以被解冻后产生自由浮动的神经球生长的NPC。在这方面，在我们的冷冻介质血清缺乏确保细胞是未分化状态保存。与众不同的条件下，神经球生产从冰冻组织显示扩张和分化率从新鲜组织生成的神经球非常媲美。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Invitrogen</td><td>11995-073</td><td>High gluclose 1X</td></tr><tr><td>Bovine calf serum supplemented</td><td>Hyclone</td><td>SH30072.03</td><td>For culture medium </td></tr><tr><td>Neurobasal-A Medium (1X), liquid</td><td>Invitrogen</td><td>1088-022</td><td></td></tr><tr><td>B27 Supplement</td><td>Invitrogen</td><td>17504-044</td><td>Supplement for Neurobasal medium</td></tr><tr><td>N2 Supplement</td><td>Invitrogen</td><td>17502-048</td><td>Supplement for Neurobasal medium</td></tr><tr><td>Trypsin (10X)</td><td>Cellgro</td><td>25-054CI</td><td>Tissue dissociation</td></tr><tr><td>Deoxyribonuclease 1 from bovine pancreas (DNase)</td><td>Sigma-Aldrich</td><td>D4527-30KU</td><td>Tissue dissociation</td></tr><tr><td>Hanks Balanced Salt Solution (HBSS) (10X)</td><td>GIBCO, by Life Technologies</td><td>14065</td><td>Cleaning tissue, washes, and freezing medium</td></tr><tr><td>Poly-L-Lysine- 500mg</td><td>Invitrogen</td><td>P2636</td><td>Substrate for adhesion of neuronal cells</td></tr><tr><td>antibiotic-antimycotic (100X), liquid</td><td>Invitrogen</td><td>15240-062</td><td>To prevent contamination</td></tr><tr><td>Large orifice pipette Tips (1-200 ul)</td><td>Fisher Scientific</td><td>02-681-141</td><td>To prevent shear stress to cells</td></tr><tr><td>Graduated pipette tips (101-1000 ul)</td><td>USA Scientific, Inc.</td><td>1111-2721</td><td></td></tr><tr><td>21 G1 precision guide needles</td><td>BD Biosciences</td><td>305165</td><td>To clean tissue</td></tr><tr><td>10 ml pipette </td><td>USA Scientific, Inc.</td><td>1071-0810</td><td>Individually wrapped</td></tr><tr><td>50 ml tubes</td><td>USA Scientific, Inc.</td><td>926-9-04</td><td></td></tr><tr><td>Single edge razor blade</td><td>Smith &amp;amp; Nephew Inc.</td><td>67-0238</td><td>To chop tissue</td></tr><tr><td>60 x 15 mm polystyrene petri dish </td><td>USA Scientific, Inc.</td><td>8609-0160</td><td>For general culture</td></tr><tr><td>100 x 15 mm polystyrene petri dish </td><td>USA Scientific, Inc.</td><td>8609-0010</td><td>For cleaning tissue</td></tr><tr><td>Cryogenic box </td><td>Nalge Nunc international</td><td>5026-1010</td><td></td></tr><tr><td>Freezing container</td><td>Nalge Nunc international</td><td>5100-0001</td><td>&quot;Mr. Frosty&quot;</td></tr><tr><td>2.0 ml cryogenic vials</td><td>Nalge Nunc international</td><td>5012-0020</td><td></td></tr><tr><td>DMSO</td><td>Fisher Scientific</td><td>D128-500</td><td>For freezing medium</td></tr><tr><td>Ethanol 200 proof</td><td>Sigma-Aldrich</td><td>E7023</td><td>For sterilizing razor blade</td></tr></tbody></table>

cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures

在这项研究中，我们勾勒出成功皮质的脑组织块冷冻保存和解冻生成神经元文化的高度浓缩的标准化协议。本议定书的冷冻介质使用的是10％的二甲基亚砜（DMSO）在汉克的缓冲盐溶液（HBSS）稀释。皮层组织块转移到含有冻结介质的离心管中，并慢慢地冻结在-1 ° C /分钟的速度控制冷冻集装箱。始终产生神经丰富的文化展出文化和神经网络的显着扩大，在第5天，10天之内迅速神经炎增长冰冻组织块解冻后的处理和分解。的星形胶质细胞标记神经胶质纤维酸性蛋白（GFAP）和神经元的标记β-微管蛋白Ⅲ类，免疫细胞化学染色显示培养的神经元和星形胶质细胞的数量高。组织块解离后的神经前体细胞培养新一代导致在迅速扩大的与众不同的条件下产生大量的神经元和星形胶质细胞的神经球，。这个简单的冷冻保存的协议，允许快速，高效，廉价的保存皮质的脑组织块，其中赠款为神经元，星形胶质细胞和神经元前体细胞培养后一代增加了灵活性。

Watch this Scientific Journal Video about Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures at JoVE.com

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

在这里，我们描述了一个高效的冷冻保存的方法和解冻皮质的脑组织块生成神经元文化的高度浓缩。这个简单的协议提供了神经元，星形胶质细胞和神经元前体细胞培养后一代的灵活性。

生成高度丰富的神经文化皮层组织块冷冻保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

cryopreservation-cortical-tissue-blocks-for-generation-highly

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

13.6K 次观看.  University of California, Irvine. 在这里，我们描述了一个高效的冷冻保存的方法和解冻皮质的脑组织块生成神经元文化的高度浓缩。这个简单的协议提供了神经元，星形胶质细胞和神经元前体细胞培养后一代的灵活性。

视频: Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

冷预处理神经保护作用的研究策略

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

科研

神经科学

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

准备在微流体装置E18条块皮质大鼠神经元

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

生物学

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法:<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养:一个快速议定书"

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

膜靶向 ca 2 + 指标对培养细胞中局部 Ca2 +信号的解剖

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

研究神经-胶质相互作用在缺血中的作用的细胞培养模型

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival. 
Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.

Here, we present a simple, potent, and versatile methodology to investigate neuronal survival upon cytotoxic stress in primary cortical neurons with cellular resolution in real time or in fixed material.

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

强大且高度可重复的皮质脑类器官生成，用于在体外模拟大脑神经元衰老

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

生成高度丰富的神经文化皮层组织块冷冻保存

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