The authors dedicate this paper in honor of Prof. Gilberto Oliveira-Castro who introduced research in intercellular communication by gap junctions in Brazil. This work was funded by Capes, CNPQ and Faperj.

<html> 1.细胞的制备<ol><li>保持胸腺上皮细胞系（IT76M1）或细胞的培养，以在培养箱进行测试（37℃/ 5％CO 2）。 </li><li>用PBS洗涤1X细胞（重复此项目3倍）。 </li><li>添加胰蛋白酶到细胞5分钟。 </li><li>添加培养基（两次胰蛋白酶的项1.3添加的量的）有10％FBS（胎牛血清）与胰蛋白酶和离心机（800×g离心5分钟）的细胞。 </li><li>算在血球细胞。 </li><li>根据细胞类型的细胞具有在彼此紧密接触，以允许耦合调节细胞的密度。注:在我们的例子中，我们使用，有35毫米的培养皿3×10 5个细胞。 </li></ol> 2，微管的制备<ol><li>如从玻璃毛细管微量（直径为1.5mm）至最终为0.2μm直径的规定拉出微量以便达到大约30兆欧的最终电阻F"&gt; 17,18。 注:另外，注射吸管就可以买到。的电阻依赖于细胞大小，例如更高的电阻微小电极将是必要的胰腺腺泡细胞，例如（100-150MΩ）19。可能出现的一个共同问题是萤光黄溶液然后可以妨碍微管，并且可能需要先过滤或离心沉淀。注射前，将微管应该在显微镜下进行分析，以检测是否存在障碍物或任何类型的中断13。微量可以通过用生理盐水溶液内的微量移液器尖端注入LY进行测试。 </li></ol> 3.测试的微<ol><li>准备在150毫摩尔/升的LiCl的萤光黄溶液（5％），并加载使用注射器微量或回填（投入它LY溶液）。 </li><li>放置微管TTE在35毫米培养皿显微注射工作站上的IT76M1细胞和淹没玻璃微尖到细胞培养基。专注于微管，并通过施加脉冲进行的染料流出试验。 </li></ol> 4.单细胞荧光黄显微注射<ol><li>聚焦显微镜正确使用高放大倍数（40X）的细胞层上面，然后慢慢放下吸管使用显微细胞。 </li><li>穿刺目标小区当尖端足够接近触摸细胞膜，并应用一个小极化脉冲引入LY到细胞中。所施加的电压将依赖于净电荷的染料被注入。或者，一些其它染料可以与本技术一起使用如表1所示。 注意:在原则上兆瓦任何亲水染料小于可以用来1KDa。然而，传输速率可以根据体重和亲水性变化。此外，U所使用的染料的nspecific转移必须进行评估。 </li><li>捕获细胞图像染料注射后3分钟，或做一个小电影时间的推移显微镜（每秒30帧）。 注:类似的方法可以在瞳等人（2015）20中可以看出。为了避免如图2由间桥通信（不完全有丝分裂），罗丹明葡聚糖（从2到10千道尔顿），它不穿过间隙连接而是通过建议间桥和某些类型的纳米管组成的共注射。 </li></ol>

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The authors have no conflicts of interest.

为了验证功能间的间隙连接的情况下，使用示踪剂，这是膜不可渗透的，尽管由间通道可渗透需要16。荧光素，所述第一荧光染料来观察细胞至细胞耦合22，是非交界膜3之间可渗透并因此被荧光黄染料15取代的。目前，要找到许多不同类型的荧光示踪剂之间的最佳选择取决于范围和实验条件。用荧光染料小区负载的过程允许形态学的评价中，单个细胞的功能和细胞之间转移的动力学速率。此外，染料显微注射可以更好地了解细胞21，23之间的间隙连接的生理作用，因为C的程度ellular通信与耦合单元的数量有关。 几个关键因素是成功获取显微注射的数据是至关重要的。正常细胞稳态和完整性必须保持，因而一个短的喷射时间（&lt;1秒）染料并用显微注射技术专长的是必要的。在得到一个更好的分辨率的拍摄图像的关键因素是有一个冷却的CCD照相机中，代替荧光过滤器和使用高NA物镜14。 1）推荐使用网格细胞培养皿来可视化的特定注入细胞，也玻璃底菜应该用于高分辨率显微镜应用;:此外，作为一些提示可能是有益的2）建议开始前，显微注射，以10-20拉针; 3）根据电极尖端的电阻可以通过触摸枪头入染料溶液是非常有用的，采用毛细作用装载微量与染料; 4）就没有必要加载吸管的全身，几微升填充尖端和2〜3毫米以上的尖端就足够了; 5）用较少的放大镜头开始实验和尝试看看吸管城墙的影子。之后，移动至吸管尽可能接近和触摸细胞之前，转移到更高放大倍数的物镜，例如40倍或更高; 6）一些团体使用气动微量。气动喷射不是最好的选择，因为它是不准确的，可能注入未知卷;用脉冲的协议可以被标准化，以注入染料的已知体积。此外，建议注入因为深度的胞质溶胶上方的膜;注射细胞核上方的膜会造成人身伤害，并影响细胞生理学。最后，是不是太扁平细胞超过平坦那些最好。 总之，这种方法是有效的通过间隙连接，但需要研究细胞间通讯专业知识/经验和良好的材料和设备，以获得高质量的数据。我们希望这篇文章和视频帮助初学者理解和执行这种技术。

间隙连接是细胞间的通道，使相邻小区间1的互通。该通信连接的两个或更多个相邻小区，其中，每一个与一个连接子或半通道有助于形成细胞间通道。在哺乳动物细胞中，所述连接子由六个连接蛋白，单体与细胞质2内4个跨膜结构域和C和N末端形成。间隙连接不仅允许离子，第二信使和小的代谢物的流动，同时也有助于在许多生理过程许多形式的蜂窝通信，诸如突触传递，心脏收缩，细胞生长和分化3，4，5，6， 7，8。除了间隙连接已与相关联许多疾病，包括癌症9，10，肌萎缩11，某些遗传性疾病和脱髓鞘疾病12。 这种类型的细胞间的串扰可以通过多种方法13，14，15，16进行评估。在本文中，我们将展示如何执行路西法黄色的单细胞显微注射可视化在活细胞通过缝隙连接处蜂窝通信。我们将讨论如何准备细胞和微量的显微的使用情况和萤光黄染料在胸腺上皮细胞系的注入。一般，此实验过程可以由平均连接的单元向装有染料的细胞进行分析。此外，这种方法可与该间隙下方分子量其他荧光染料被用于路口切断这大约是1000道尔顿。

<table><tbody><tr><td>Lucifer yellow</td><td>Sigma</td><td>L0259</td><td></td></tr><tr><td>Lithium Chloride</td><td>Sigma</td><td>L4408</td><td></td></tr><tr><td>PBS tablets</td><td>Sigma&amp;nbsp;</td><td>P4417</td><td></td></tr><tr><td>RPMI</td><td>Sigma</td><td>R4130</td><td></td></tr><tr><td>Bovine fetal serum</td><td>Cultilab</td><td></td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T4799</td><td></td></tr><tr><td>vibration-insulated table&amp;nbsp;</td><td>Newport</td><td>VH3036W-OPT</td><td>A vibration-insulated table is needed to protect the experiments from vibration and avoid cell damage</td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MMO-203</td><td>This equipment allows precision adjustments of the micropipette, which is needed for cell micro injection.</td></tr><tr><td>Current Generator&amp;nbsp;</td><td>Digitimer</td><td>DS2</td><td>To produce the dye flow through the micropipette, a current below one nano ampere was given using a current generator with an electrode inside the micropipette or an amplifier which has a capacitance compensation circuit (old electrometer) or current injection functions of new patch clamp amplifiers, and the ground wire submersed in the plate dish. Alternatively, the dye can be injected by a pneumatic microinjector, following the factory recommendations.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr></tbody></table>

single-cell microinjection for cell communication analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

胸腺上皮细胞系IT-76MI用于通过缝隙连接来评估染料耦合，因为这些细胞被描述来表达连接蛋白43 21形成功能性缝隙连接。图1示出了当在吸液管的尖端下方的一个小区施加萤光黄的注入。几分钟后，连接的电池变成指示荧光染料通过间隙连接的扩散荧光（星号）。细胞和时间成为荧光的数量直接与这些细胞中的蜂窝通信的程度相关联。 图2示出LY的胸腺上皮细胞的注射和插入件（插入d）示LY和罗丹明葡聚糖（10KDa）的共注射。如预期的那样LY传递到相邻小区，尽管若丹明葡聚糖不其分子量高，因为。此外，GJ阻塞的存在（插入f）中，辛醇，一个BLOcked染料到周围细胞的通过。另外，可以评估一个特定的细胞或药物对GJ的功能的影响的耦合度。图3A示出LY的了TEC细胞有或无地塞米松的注入。然后100细胞注射，在地塞米松存在下和1或2个细胞进行通信的百分率是相对于无药物对照相似。然而相比，控制时3或4个细胞进行通信的数量较高。五个或6个细胞和7或8细胞与地塞米松存在下GJ通信中的控制，没有观察到，因此表明塞米松增加耦合了TEC细胞的程度。 <img alt="图1" src="/files/ftp_upload/50836/50836fig1.jpg" /> 图1:荧光黄注射液在IT-76MI细胞。 （A）中的微量接近细胞膜。 （B </strong&gt;的）的吸移管通过荧光显微镜观察。生成（C）的测试脉冲来验证电极是否实际注入的染料。 （D）的移液器触摸细胞膜和它被充电与萤光黄染料。 （E）的充电的电池允许染料通过间隙连接传递到至少五个相邻小区（由箭头指示），X20。 F）数码变焦做是为了能够更好地可视化。星号指出的对比度相显微镜（图1A）中加入该细胞。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/50836/50836fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/50836/50836fig2.jpg" /> 图2:荧光黄注射胸腺上皮细胞（TEC）。相衬和荧光显微镜。 （A </strong&gt;的信息和B）人类胸腺上皮细胞。 （C和D）胸腺细胞护士分别为（E和F）的小鼠胸腺上皮细胞系。 （D）中插入表示罗丹明-葡聚糖10KDa的注射（通过间隙连接不透）之后的相同的细胞（箭头）。在（E）和（F）的插入件显示没有在TEC符合辛醇为1mM（间隙连接阻断剂）10分钟预处理染料的转移。在所有面板星号标记注射细胞。 （AF）×200从阿尔维斯等人转载。 ，1995年5。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/50836/50836fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/50836/50836fig3.jpg" /> 图3:缝隙连接交流沟通n的大鼠上皮细胞系增加了地塞米松。 TEC分别用1μM地塞米松治疗，和耦合程度是由荧光黄染料转移测定法进行评估。 （A）的显微字段（相衬和荧光，分别在左，右面板）描绘了注射细胞和那些当LY注射（倍率320X），该偶合。在所有面板星号标记注射细胞。 （B）的直方图表示控制和地塞米松处理的细胞的耦合的图案。该分析包括每组100显微注射的。从阿尔维斯等人，2000年23再现。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/50836/50836fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 染料 </td><td> MW </s仲&gt; </td><td> 激发/发射 </td></tr><tr><td>羟基香豆素羧酸</td><td> 206 </td><td> 488分之386 </td></tr><tr><td>钙黄绿蓝</td><td> 321 </td><td>四百四十九分之三百六十○ </td></tr><tr><td> 4&#39;，6-二脒基-2-苯基二盐酸盐</td><td> 279种</td><td> 461分之358 </td></tr><tr><td>羧基</td><td> 376 </td><td> 517分之492 </td></tr><tr><td>碘化乙锭（溴） </td><td> 314 </td><td>六百〇五分之五百一十八</td></tr><tr><td>荧光黄CH </td><td> 443 </td><td> 536分之428 </td></tr><tr><td>的Alexa Fluor 488 </td><td> 570.5 </td><td> 519分之495 </td></tr><tr><td>钙黄绿素</td><td> 622 </td><td> 517分之494 </td></tr><tr><td>碘化</td><td> 414 </td><td> 617分之535 </td></tr></tbody></table> 表1:染料目前用于显微注射实验。在这里，一些用于与分子量和激发/发射波长染料。

Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

Single-cell Microinjection for Cell Communication Analysis

在这里，我们将介绍如何执行路西法黄色的单细胞显微注射可视化在活细胞通过缝隙连接处蜂窝通信，并提供一些有用的提示。我们希望本文可以帮助大家来评估细胞耦合程度由于功能间隙连接。这里所描述的一切可能的，在原则上，适合于与低于1,000道尔顿分子量其它荧光染料。

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

single-cell-microinjection-for-cell-communication-analysis

Universidad San Sebastian

Research

JoVE Journal

Biology

11.2K 次观看.  Oswaldo Cruz Foundation. 在这里，我们将介绍如何执行路西法黄色的单细胞显微注射可视化在活细胞通过缝隙连接处蜂窝通信，并提供一些有用的提示。我们希望本文可以帮助大家来评估细胞耦合程度由于功能间隙连接。这里所描述的一切可能的，在原则上，适合于与低于1,000道尔顿分子量其它荧光染料。 

视频: Single-cell Microinjection for Cell Communication Analysis

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

单细胞的转录组学分析

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

科研

神经科学

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

生物工程

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

游离成年哺乳动物元DNA的细胞核内内微量注射

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

生物学

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

使用机器人微注射操作脑片中单神经干细胞和神经元

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

使用微注射在人赫拉细胞中分体血球定位分析

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Microinjection of Drosophila Nurse Cells: A Method of Intracellular Delivery

Source: Catrina, I. E., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/58545/" target="_blank">Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers</a>. J. Vis. Exp. (2019).
Due to their large size and relatively simple organization, Drosophila nurse cells are ideally suited for live cell imaging studies. This video describes a microinjection method for exogenous compounds delivery into the cells, and the featured protocol demonstrates the procedure with fluorescent oligonucleotide probes called molecular beacons (MBs) used to visualize endogenous mRNA transcripts.

果蝇护士细胞的显微注射:一种细胞内递送方法

Source: Catrina, I. E., et al. Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers. J. Vis. Exp. (2019).
Due to their ...