该实验室已经由加拿大健康研究机构，自然科学和工程研究理事会，加拿大，和加拿大（MSSC）多发性硬化症协会的支持。商品及服务税是一个从MSSC博士助学金的收件人。我们非常感谢博士琼博格斯（多伦多病童医院）这个手稿中许多有益的讨论和意见。 Biotium公司额外NucView 488 caspase - 3的厚礼，我们非常感谢。

1。解冻和培养细胞<ol><li>获取股票冻结永生N19的少突胶质细胞从长期液氮存储。 </li><li>浸入小瓶含有细胞在37℃水浴，直到完全解冻的细胞悬液。 </li><li>添加7毫升（贝科的改良Eagle培养基）的DMEM高糖补充与10％FBS（胎牛血清）和1％青霉素/链霉素10厘米的培养板。 </li><li>添加下拉明智的板块，并轻轻搅拌和岩石的Petri板均匀分散的细胞的细胞悬浮。 </li><li>在34 ° C / 5％CO 2培养箱培养细胞。 </li><li> 4小时后，从板的吸媒体删除任何剩余的DMSO（二甲基亚砜），已作为冷冻保护剂的使用，并取代与新媒体（7毫升的DMEM高糖媒体与10％胎牛血清和1％青霉素/链霉素） 。 </li></ol> 2。传代细胞<ol><li>在70-80％合流（4-7天增长），从细胞吸媒体。 </li><li>加入1毫升的0.25％胰蛋白酶板块。移液器胰蛋白酶分离细胞（约5分钟）。 </li><li>实验条件，并通过一个额外的10厘米板未来的实验在细胞密度不小于0.1 × 10 6细胞/毫升适当稀释。 </li></ol> 注意：您应该通过使用实验前解冻后的细胞至少两次。 3。细胞计数和电镀<ol><li>对活细胞成像板细胞，trypsinize如前所述。 </li><li>取出约30μL细胞计数使用一个血球。 </li><li>将在6孔板无涂层的玻璃盖玻片。以及细胞密度为0.1 × 10 6细胞/ ml与10％FBS和1％青霉素/链霉素，在34 ° C / 5％CO 2补充，在2毫升苯酚的DMEM高糖媒体。 </li><li>允许细胞生长过夜（16-20小时）在34 ° C / 5的转染前2％的二氧化碳。 </li></ol> 4。转<ol><li>将100μL的无血清培养基，0.5-4微克纯化的质粒DNA，和4μL，FuGENE HD（罗氏）。涡流轻轻混合。 </li><li>涡再次简要，并允许在室温下5分钟到复杂的DNA，然后添加FuGENE HD DNA混合物直接向培养细胞。倾斜板，轻轻地混合。 </li><li>文化的细胞在34 ° C / 5％的CO 2的待遇或实验前一个额外的48小时。 </li></ol> 5。准备活细胞成像细胞（LCI） <ol><li>对LCI Chamlide活细胞的仪器控制箱打开至少1小时前你要开始实验。控制箱还规定的国际狮子会Chamlide内的温度和湿度，调节流预混后的5％CO 2。 </li></ol> 注：这是明智的打开控制箱3小时实验开始前，以确保整个舞台达到34 ° C。这一步将减少焦点漂移，这是fluxulation和热膨胀的金属阶段引起的，因为它温暖，在成像。 <ol start="2"><li>工作在流罩，喷Chamlide磁型文化室，镊子，用70％乙醇，并让他们干5分钟。 </li><li>从培养箱中取出细胞，并确认它们是健康的的。他们应该会出现贴壁，并与众多的膜过程玻璃盖玻片上的传播。强调转的细胞并不适合用于实验，将有一个过程扩展的数量减少，而且往往有一个不规则形的细胞核。 </li><li> 6孔板倾斜，并取出镊子盖玻片。快速放置盖玻片细胞侧成文化室底板。不要让盖玻片干。 </li><li>将磁主要身体的文化室，并添加500μL媒体从原来的6孔盘到盖玻片上方。重新使用这种媒体将减少对环境变化所造成的细胞应力的量，也可以用于评估外分泌的因素。 </li><li>广场文化室的玻璃盖。 </li><li>使用KimWipe喷用70％乙醇，以去除任何残留物质从底部的盖玻片一个，这会干扰用显微镜。 </li><li>文化室放置在34 ° C / 5％ 二氧化碳培养箱中培养30分钟。这一步骤将确保分庭本身预热至34 ° C至减少盖玻片转移金属升温。 </li></ol> 6。显微镜设置图片在这里获得使用一个自定义的中继镜头和排放过滤器墨盒加载多个轮子的车轮罩（法定人数科技公司，G徕卡DMIRE2倒置显微镜uelph，ON）。 <ol><li>明场 - 灯设置为2.5 V和240毫秒的曝光时间。 </li><li>在140红色荧光蛋白（RFP） - 增益设置为200毫秒。 </li><li>在140绿色荧光蛋白（GFP） - 增益设置为500毫秒。 </li><li>我们的显微镜调光灯控光对细胞的数量，而不是一个旋转的磁盘。我们光与运行我们的实验中，最大的灯强度的90％。 </li></ol> 7。细胞治疗与细胞凋亡诱导剂和NucView 488 Caspase - 3的基板<ol><li>重要的是要提前准备凋亡诱导剂的大型股，并冻结他们在等分，使浓度将之间的实验一致。 </li><li> NucView 488基板是光敏感。准备分装15μL，以减少冻结/解冻，并储存在-20 °铝箔覆盖的彗星管。用尽可能低的室温照明工作，以减少暴露的NucVIEW 488基板的光，在使用前。 </li><li>迅速从孵化器中检索的文化室，使细胞不暴露在温度降低。放置到显微镜的环境室文化室，并用夹具保持移动的文化室。在这一点上，你也应该昏暗的房间的灯。 </li><li> 5％的CO 2预混罐，打开双调节。 </li><li>使用10倍的目标，注重发现细胞在计算机上使用明场显微镜的监视器注意 ：你想使用所需的放大倍率客观上最大的数值孔径，以减少曝光时间。 </li><li>提供诱导细胞凋亡的细胞（在我们的案例80毫米钾，或100毫米谷氨酸），可以使用下列方法之一。我们将使用80毫米钾为例交付，使用作为一个典型的文化传媒的10倍浓缩溶解氯化钾。 <ol><li> 媒体B股Ÿ慢和局部灌注： - 使用含80毫米钾的新媒体文化室交流媒体蠕动泵。 - 管端将提供一个80毫米钾溶解在酚无DMEM的股票，1​​0毫升和油管的另一端，将删除从会议厅的媒体。 - 你想媒体至少10倍的交流，以确保在会议厅内离开媒体正确的钾浓度。 -交换媒体后，加3μLNucView 488基板，在会议厅内的媒体。吸管组合。 </li><li> 80毫米K +和NucView 488基板媒体在文化室直接加入 ： - 准备了80毫米钾溶解在酚无DMEM 10倍原液。 -加入3μLNucView 488基板50μL，10倍80毫米钾。吸管组合。添加500μL，苯酚的DMEM在文化室。 注：此方法是首选，如果你有兴趣，在保持或评估增长或其他分泌的因素可能存在于原始媒体。我们发现，只要持续36小时的实验 NucView 488基板是在细胞培养稳定</li></ol></li></ol> 8。活细胞成像<ol><li>点击显示转染细胞上的红色通道。 </li><li>选择并保存周围十几帧，那里有几个转染细胞，并保存这些“XY阶段分”。根据计算机的内存量，则可能是有限的阶段点的数量，你将能够获得。 </li><li>在这些保存的阶段有显微镜捕捉的图像（明场，红色通道和绿色通道）点每6分钟。 </li><li>任何绿色的染色，可见在实验的初期阶段，可能表明通常是由于细胞转染和/或环境压力，已经发生凋亡。 Apopt将在稍后时间点在实验中发现尘埃沉着病是由于实验治疗。 </li></ol> 9。统计分析<ol><li>对于每一个实验中，我们计划在显微镜获得多重的XY点收集一套有效的大型数据。每个实验进行重复或一式三份，数据均来自独立的实验在不同的日子进行编译。我们从每个数据集，分析15视场和比较caspase阴性的细胞凋亡阳性细胞（细胞总数在实地查看/凋亡阳性细胞总数）的比例。 </li><li>从每一个数据集记录的测量组合成一个较大的样本集，然后另一个使用ANOVA表（P = 0.05）相比。我们显示平均每个实验的标准误差（SEM）的，然后比较在手段上的分歧，通过执行一个杜克手段的对比测试（P = 0.05），以确定WHICH治疗显著彼此不同。 </li></ol> 10。代表性的成果我们已经描述了一个实验来说明如何NucView 488基板可以显示N19 - OLG细胞培养细胞凋亡率增加，下面的治疗与高外钾离子浓度。 N19细胞转染与RFP，要么与3μL，NucView 488基板（控制），或3μLNucView 488基板和80毫米的治疗[K +]（治疗） 。细胞进行了监测和图像均超过12 h时间当然，这是足够的研究涉及神经系统的侮辱（ 图1A）收购。在控制条件下，我们没有观察大量的细胞凋亡的80毫米相比，[K +]对待文化（哈希盒），这表明约45％的细胞死亡后12小时（ 图 1B ）。背景绿色信号观察在控制条件下，d表示接受没有增加细胞外钾的细胞凋亡的细胞，。几乎没有控制实验展览由12 h时间点凋亡细胞，但在其他情况下的实验，可能需要运行的时间更长。获得10倍的目标。酒吧= 100微米。 <img alt="图1a" src="/files/ftp_upload/3422/3422fig1.jpg" /> <img alt="图1b" src="/files/ftp_upload/3422/3422fig1b.jpg" /> 图1。 （A）12小时的时间N19 OLG文化课程实验48 h后转染，表达RFP NucView 488基板（绿色通道）3μLMBP（红色通道） 。文化是可以治疗的终浓度为80毫米[K +]（左侧面板），或无治疗作为对照（右图）。图像获得6分钟的间隔，并显著裂解的caspase - 3激活（绿色信号）可观察（二）D在对待文化与80毫米[K +在细胞核（哈希箱），比控制实验。裂解caspase - 3的细胞（细胞总数除以视野计算的百分比凋亡阳性细胞总数）。在控制条件相比，我们观察到约45％细胞死亡后，K +处理12小时 SVIDEO 1一个12小时的时间N19 - OLG文化课程实验，48小时后转染表达RFP 3μLNucView 488基板（绿色通道）MBP（红色通道），与明场图像和三路沿合并后的形象，与80毫米以下的治疗[K +] 。 2。SVIDEO一个12小时的时间N19 - OLG文化课程实验48 h后转染表达与3μLNucView 488基板（绿色通道）RFP - MBP（红色通道），随着明场成像ES和三路合并后的图像。无治疗应用的文化和N19 - OLGs通过显微镜领域的迁移，可以看出在80毫米治疗的细胞培养[K +]（比较SVIDEO 1）。

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作者有没有利益冲突披露。

虽然这不是唯一的荧光产品可用于检测细胞凋亡，有几个显着的优势， 使用NucView 488基板。的主要好处之一是能够实时跟踪在活细胞的凋亡，而大多数的替代产品需要细胞裂解或细胞的通透性差。其他好处包括caspase - 3的识别，细胞的通透性高，低毒性，并与细胞凋亡的进展无干扰的高灵敏度。基板还具有低背景荧光，直到它被切割，进入细胞核，从而消除背景荧光。 caspase - 3的识别序列包含3负电荷的DNA结合的染料有一个正电荷。 DEVD - NucView 488分子，因而具有净负电荷，从而阻止细胞不活跃的caspase的活化和染料结合到DNA 。 马intaining实验之间的一致性是需要能够进行有意义的比较借鉴之间复制。保持一致的主要参数之一是细胞密度，因为它影响的转染率。获得高转染率在永生N19 OLG细胞培养超过其他常见的细胞株，如HeLa细胞或HEK293困难，是在我们的经验，12，13，高度依赖于密度。我们对这些细胞的最佳转染效率都得到Fugene HD（罗氏），通常约15％，但可以达到30％以上，根据构造。仔细的细胞计数，将有助于取得一致的转染率。同样重要的是揭露，从不同的治疗环境压力相同程度的细胞，尤其是当测量凋亡率。具体来说，时间的长度，细胞暴露转染试剂，或光线照射量，是重要的环境变量ables考虑，并应保持在实验常数。对于我们的应用程序中，我们发现，搜集了大量的字段的视图提供一个足够大的样本大小，使统计显著比较[同上] 。

特定的试剂和设备表<table border="1"><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> NucView 488 Caspase - 3的活细胞检测试剂盒</td><td> Biotium公司</td><td> 30029 </td></tr><tr><td> FuGENE高清转染试剂</td><td>罗氏公司</td><td> 04709705001 </td></tr><tr><td>贝科的修改鹰中等</td><td> Gibco公司</td><td> 31053-028 </td></tr><tr><td> 0.25％胰蛋白酶</td><td> Gibco公司</td><td> 15050-065 </td></tr><tr><td>胎牛血清</td><td> Gibco公司</td><td> 12483-020 </td></tr><tr><td>青霉素/链霉素</td><td> Gibco公司</td><td> 15140122 </td></tr><tr><td> ＃1.5-25毫米玻璃盖玻片</td><td>华纳仪器</td><td> 64-0715 </td></tr><tr><td> Chamlide中巴磁 文化室为25毫米盖玻片</td&gt; <td>法定人数技术</td><td> CM - B - 40 </td></tr><tr><td> Cellstart组织文化10厘米的菜</td><td> VWR </td><td> 82050-576 </td></tr><tr><td> BD猎鹰6孔组织培养皿</td><td> VWR </td><td> CA62406 - 161 </td></tr></table>

monitoring cleaved caspase-3 activity and apoptosis of immortalized oligodendroglial cells using live-cell imaging and cleaveable fluorogenic-dye substrates following potassium-induced membrane depolarization

中枢神经系统可经验的强调和神经系统的侮辱，它可以有许多不利影响，减少神经元的人口和功能，最终导致数量。受损的轴突可以释放分子，包括钾或到细胞外基质，这反过来，可以产生进一步的侮辱和伤害，包括星形细胞和少突胶质细胞8，16支持神经胶质细胞谷氨酸兴奋。如果侮辱仍然存在，细胞会进行程序性细胞死亡（凋亡），这是规范和激活一些行之有效的信号转导级联 14 。细胞凋亡和组织坏死后可出现脑外伤，脑缺血和癫痫发作。细胞凋亡调控的一个经典的例子就是家庭的依赖半胱氨酸天冬氨酸指示蛋白酶，或凋亡。包括caspases的活性蛋白酶也被牵连在慢性神经细胞死亡退行性疾病包括阿尔茨海默氏症，亨廷顿氏，和多发性硬化症4，14，3，11，7。 在这个协议中，我们描述 NucView 488 caspase - 3的基板来衡量的caspase - 3的永生N19，少突胶质细胞（OLG）细胞培养15日，5介导的细胞凋亡率，暴露于高浓度，如不同外讲钾或谷氨酸。有条件永生N19 OLG细胞株（代表O2A祖）获得的安东尼Campagnoni博士（加州大学洛杉矶分校塞梅尔神经科学研究所），15，5，和先前已用于研究髓鞘基因表达和信号转导的领先的分子机制OLG分化（如6，10）。我们发现这种细胞系转染外源性髓鞘碱性蛋白（MBP）的强劲构造融合RFP或GFP（红色或绿色荧光蛋白）13 ，12。在这里，N19 - OLG细胞培养治疗，为80毫米氯化钾或100毫米的谷氨酸钠模仿轴突漏到细胞外基质诱导细胞凋亡9。我们用一个双功能的caspase - 3基板包含一个DEVD（ASP - GLU - VAL - ASP）Caspase - 3识别亚基与DNA结合染料2。基板迅速进入细胞质中，它是由细胞内的caspase - 3裂解。 NucView 488染料，被释放并进入细胞核，它在488 nm的结合DNA和绿色荧光，信号凋亡。 NucView 488 Caspase - 3底物允许使用活细胞成像实时1，10。在这段视频中，我们还描述了不朽的N19 - OLG细胞的培养和转染，以及活细胞成像技术。

Watch this Scientific Journal Video about Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates F

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

活细胞成像的caspase - 3介导的细胞凋亡，永生N19少突胶质细胞培养使用<em&gt; NucView</em&gt; 488的caspase - 3基板。这种技术适用于多种类型的细胞和组织细胞程序化死亡实时检测。

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

monitoring-cleaved-caspase-3-activity-apoptosis-immortalized

Research

JoVE Journal

Neuroscience

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

15.8K 次观看.  University of Guelph. 活细胞成像的caspase - 3介导的细胞凋亡，永生N19少突胶质细胞培养使用 NucView 488的caspase - 3基板。这种技术适用于多种类型的细胞和组织细胞程序化死亡实时检测。

视频: Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

科研

神经科学

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala &#8211; peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 &#176;C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

Caspase-3活性在鼠杏仁核测量用荧光分析法心肌梗死后

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

利用 SPM 软件分析颅内脑电图数据的神经活动和连接性

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

神经元发育过程中的 ROS 活细胞成像

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many neurodegenerative diseases. Given some axons' elaborate architecture and extreme length, it is not surprising that mitochondria in axons can experience different environments compared to their cell body counterparts. Interestingly, dysfunction of axonal mitochondria often precedes effects on the cell body. To model axonal mitochondrial dysfunction in vitro, microfluidic devices allow treatment of axonal mitochondria without affecting the somal mitochondria. The fluidic pressure gradient in these chambers prevents diffusion of molecules against the gradient, thus allowing for analysis of mitochondrial properties in response to local pharmacological challenges within axons. The current protocol describes the seeding of dissociated hippocampal neurons in microfluidic devices, staining with a membrane-potential sensitive dye, treatment with a mitochondrial toxin, and the subsequent microscopic analysis. This versatile method to study axonal biology can be applied to many pharmacological perturbations and imaging readouts, and is suitable for several neuronal subtypes.

The present protocol describes the seeding and staining of neuronal mitochondria in microfluidic chambers. The fluidic pressure gradient in these chambers allows for the selective treatment of mitochondria in axons to analyze their properties in response to pharmacological challenges without affecting the cell body compartment.

微流体辅助轴突线粒体选择性去极化

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

人小胶质细胞样细胞：使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

使用 STED 显微镜揭示神经元中的线粒体超微结构

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

作者聚焦：解码线粒体衰老 

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

使用活细胞STED成像可视化神经元细胞模型中的线粒体内膜超微结构

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...