This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).

动物实验已通过了伯尔尼州Kantonstierarzt的地方委员会，KantonalerVeterinärdienst伯尔尼（BE85 / 15）。 1. 爪蟾卵母的制备<ol><li>保持在12小时/ 12小时光照/黑暗中水的循环是严格保持在20℃，蛙（ 爪蟾 ）。 </li><li>从雌蛙9取出卵巢的叶。 注意:凸起的除去是用于再生的刺激，并经常在卵母细胞的质量与手术改善。卵母细胞被卵黄层，卵泡细胞的层，并且含有血管21结缔组织覆盖。整个结构被称为"毛囊"。 </li><li>放置被密集用含有无菌巴特的盐7（MBS）的卵母细胞补充有青霉素和链霉素（88毫摩尔NaCl，1mM的氯化钾，2.4毫毛囊卵巢的裂片的NaHCO 3，0.82毫硫酸镁 ，0.41毫氯化钙 ，0.33毫米的Ca（NO 3）2，10毫4-（2-羟乙基）-1-哌嗪乙磺酸; HEPES（pH值7.5，氢氧化钠），100微克/毫升青霉素和100微克/ mL链霉素）。 </li><li>单出阶段V-VI 21卵泡。握住钳子卵巢叶。降低铂环（ 图1）在卵泡并轻轻撤回循环扰乱含有从卵巢组织卵泡结缔组织。 注:第五阶段-Ⅵ21卵泡的特征在于它们的尺寸（1.0 - 1.2毫米直径），并通过暗着色动物半球和淡黄色植物性半球之间的良好的对比度。 </li><li>通过塑料巴斯德吸管（切回1.5mm的开口直径）的装置仅传送看起来健康的卵泡（ 即完美的球形，并用完整的色素沉着）到35毫米直径的培养皿。 </li></ol>注:此处描述的显微注射系统是从由Kressmann和Birnstiel 22日报道的。 <ol><li>准备从相应的cDNA 通过体外转录A受体，增加了一个多聚（A +）尾和RNA定量的用凝胶电泳23编码的α4β2δGABA的亚基的cRNA。 </li><li>从使用微量牵拉硼硅玻璃毛细管（1.0外径（OD），0.58内径（ID），100毫米长）制备显微注射移液器。断的玻璃毛细管的尖端使用显微和微丝创建12的顶端直径，在显微镜下 - 15微米与斜尖。 </li><li>回填用10毫升的注射器石蜡油显微注射吸管，然后装入显微注射吸管到一个自制的MICRoinjection装置（ 图2）。 注:自制液压油喷射装置包括一个脚踏开关控制烧烤电机。一个附加的开关改变马达的转动模式。该电机驱动的推进或缩回10μL玻璃注射器的柱塞螺旋测微计（0.5毫米/转）。注射是在约3 rpm的速度和0.5转弯对应于50 NL注射到毛囊。注射器的前端连接到本身由非常短的一块Tygon管的连接到注射针厚壁聚四氟乙烯管材。加样毛细管是由一个由显微控制的钻夹头保持。整个系统充满了石蜡油。任何气泡应该避免，因为它们会损害喷射系统。设置点亮了冷光源。立体显微镜需要光学控制。卵泡在冷光可视化与stereomicroscope需要（40X放大倍率）。注射设定的性能由注射放射性示踪剂进入爪蟾卵母测试。 45卷 - 55 NL应该每次注射交付。 </li><li>使用移液管，用无菌塑料尖端，通过将无菌水滴到耐湿热塑性（2×2厘米）的清洁内部测试设置。沉浸注射吸管到该液滴的前端，然后转动电机缩回柱塞（注射吸管内部负压力）。 注:水应该进入吸管和形成与油中的可视界面。 </li><li>退离液滴的吸管尖和施加正压到注射移液管的内部。水滴应该形成在注射吸管尖。 </li><li>粘到PE的底部:通过衬里它们与植物性磁极在尼龙网的空隙（0.8毫米G）的朝上准备毛囊的喷射三菜（60 MM）覆盖着MBS。 </li><li>使用移液管，用无菌塑料尖端到热塑性的清洁内部分配mRNA的2微升。通过施加负压到注射移液管的内部取的mRNA向上进入注射针。 </li><li>注射针在使用显微单个毛囊的位置。 </li><li>插入注射针进入植物极的中心与通过施加正压力，以移液管的内部注入mRNA的50 NL以0.6微升/分钟的流速。从毛囊取出注射枪头，以避免逃跑的mRNA前10秒 - 等待5。 注:基因应注入的植物（淡黄）极，以避免注射到细胞核中，它位于在动物半球。 </li><li>传输所注入的毛囊填充有2毫升的MBS的新的培养皿（35毫米）。放置盘成酒冷却器设定为18℃。孵育1注入的卵泡 -记录前7天，这取决于新表达的蛋白的身份。 </li></ol> 3.卵泡剥离（图3） 注:如上所述，由卵黄层，卵泡细胞，结缔组织，其中包含血管24所覆盖的卵母细胞，被称为"毛囊"。除了卵黄层，它提供机械稳定性不妨碍到细胞表面的解决方案的进入所有的层，必须电生理实验之前被移除。周围无细胞层卵泡先前已称为"洁净"卵母细胞25。此步骤通常在同一天的电生理实验进行。 <ol><li>转移10注入卵泡到含有0.5毫升的MBS，1毫克/毫升胶原酶和0.1mg / ml胰蛋白酶抑制剂的硼硅玻璃管。浸入管保持在36℃水浴。孵育20分钟卵泡和动摇管偶尔。 </li><li>通过将它们转移到含有1毫升的MBS在RT第二管冲洗卵泡。让他们在管约10秒。 </li><li>转移卵泡到包含含有4mM乙二醇双（2-氨基乙醚）加倍浓缩的MBS 0.5mL的第三管- N，N，N&#39;，N&#39;-四乙酸（EGTA），并孵育他们在4分钟RT。偶尔摇管。 注:高渗溶液（加倍浓缩MBS）用于诱导卵泡细胞层内的卵母细胞，从而分离从卵母细胞滤泡细胞层的收缩率加入EGTA以抑制胶原酶活性。 </li><li>通过将它们转移到含有1毫升的MBS在RT第二管冲洗卵泡。让他们在管约10秒。 </li><li>转移的卵母细胞，以含2毫升的MBS培养皿（35毫米直径）。在立体显微镜，通过分开毛囊外封套简单地推裸卵母细胞离开使用铂环。 注:卵母细胞的外信封牢牢粘在培养皿，并可以从卵母细胞容易地分离。有些卵母细胞会自发地失去他们的外封套，将被回收裸卵。此过程导致用于保存的卵母细胞的球形外观相对稳定的制剂。 </li></ol>周围的卵母细胞4.快速变化的解决方案<ol><li>进行电压钳实验中，如所描述9,26。 </li><li>通过根据需要由试验转动灌注开关改变重力供给灌注系统的解决方案。 <ol><li>用玻璃毛细管1.35毫米，口，其中放置约0.4mm从卵母细胞表面的内径施加在6毫升/分钟的灌注溶液，以允许围绕卵母细胞的激动剂浓度的快速变化。 注意:变化率一直此前估计ð在小于0.5秒27的70％。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这篇文章中描述的方法，从那些传统使用的7-9偏离。它是标准的对卵巢的裂片暴露于1至2小时胶原酶处理8;隔离损坏，裸卵;与使用商业注射装置的mRNA注入它们。此经典程序具有以下缺点:1）卵母细胞很可能是由长曝光于高浓度的胶原酶被损坏。 2）不稳定裸露的卵母细胞必须被存储，直到实验。 3）裸卵更有可能比注射过程中的卵泡挨。商业喷射设备使用大直径（约20微米）注射?...

爪蟾卵母被广泛用作表达系统（参考文献2 - 3，和其中的参考文献）。它们能够正确地组装和整合功能活性多亚基蛋白到其质膜。使用该系统，有可能在功能调查膜蛋白单独使用或与其它蛋白质组合，为了研究突变，嵌合的，或连接在一起的蛋白质的性质，以及筛选潜在药物。 使用的卵母细胞比其它异源表达系统的优点包括巨细胞的简单处理，表达外源遗传信息，卵母细胞的环境的通过浴灌注装置的简单的控制的细胞的比例高，和膜电位的控制。 该表达系统的缺点是在许多实验室17-20中观察到的季节变化。这样做的原因变异目前尚不清楚。此外，卵母细胞的质量经常观察到强烈变化。传统方法7-9包括卵巢瓣的隔离，卵巢瓣的暴露于胶原酶一些小时，裸露的卵母细胞的选择，以及卵母细胞注射。在这里，一些替代方案，快速程序报道，使我们能够用这个表达系统工作超过30年，在卵母细胞质量无季节变化和变化不大。 此处描述了用于卵母细胞的分离，微注射用的cRNA，并除去滤泡细胞层的改性，改进的方法，可用于选择的任何蛋白在非洲爪蟾卵母细胞中的表达。周围的卵母细胞的培养基中快速解的变化非常简单的方法可以应用于任何配体门控离子通道和载流子的的研究。

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xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments

非洲爪蟾卵母细胞作为异源表达系统用于蛋白质，首先由Gurdon 等人所述。 1并已发现以来被广泛使用（参考文献2 - 3，和其中的参考文献）。这使得外国通道表达卵母细胞有吸引力的一个特点是内生的离子通道4的丰度差。这种表达系统已被证明对于许多蛋白质，其中配体门控离子通道的特征是有用的。 GABA A受体在爪蟾卵母细胞和其功能特性的表达被这里描述，包括卵母细胞，用的cRNA，去除滤泡细胞层，并在电生理实验快溶液变更显微注射的隔离。的过程被在这个实验室5,6-优化，从常规使用7-9的那些偏离。传统上，裸卵准备用在RT延长胶原酶处理卵巢裂片的，并且这些裸露的卵母细胞显微注射用的mRNA。采用优化方法，多元膜蛋白已表达并研究了该系统中，如重组GABA A受体10-12，人重组氯通道13，锥虫钾通道14，和一个肌醇转运15,16。 这里详细描述的方法可以应用于在爪蟾卵母选择的任何蛋白质的表达，并迅速解变化可以用来研究其它配体门控离子通道。

爪蟾卵母细胞进行机械挑出使用铂环（ 图1）。的卵母细胞显微注射用的mRNA编码GABA A受体亚单位α4，β2，δ，0.5:0.5:2.5飞摩尔/卵母细胞（ 图2）。后4天，滤泡细胞层除去（ 图3）。卵母细胞电压钳制在-80毫伏和暴露于1μM的3α，21-二羟基5α孕-20-酮（THDOC），一种有效的正变构调节剂的存在增加γ氨基丁酸?...

Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

爪蟾卵母:显微注射方法优化，滤泡细胞层的去除，并在电生理实验快速变化的解决方案

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

xenopus-oocytes-optimized-methods-for-microinjection-removal

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

20.1K 次观看.  University of Bern. Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented. 

视频: Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

小鼠卵母细胞的细胞核移植到

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

科研

生物学

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

补丁钳爪蟾卵母细胞中表达的离子通道

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

双层弹性性能的监测变化的单分子方法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

施肥爪蟾卵母细胞使用主机传输方法

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

酵母发光计量和<em&gt;爪蟾</emTRPV4的分子Mechanosensitivity的&gt;卵母细胞电生理检查

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

爪蟾卵母:显微注射方法优化，滤泡细胞层的去除，并在电生理实验快速变化的解决方案

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