We thank Lowell Cochran for expert videography assistance. The financial assistance of the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Breast Cancer Research Alliance, the Ontario Centers of Excellence, the Breast Cancer Action Kingston and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. SG was the recipient of an NSERC studentship. MG was supported by a postdoctoral fellowship from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#8217;s University.

1，电镀的电穿孔室中的电解槽<ol><li>在层流罩，使用无菌技术如常trypsinize细胞。 注意:通过离心来消除胰蛋白酶的所有痕迹，因为它们可能会妨碍细胞的玻璃板上散布，粘着和间隙连接从而形成是非常重要的。 </li><li>吸管将1ml细胞悬液中提供的原位电穿孔仪（ 图1），并将其放置在37℃， 二氧化碳培养箱无菌电室的。 注:细胞粘附可以通过电镀于纤连蛋白，胶原蛋白，多聚赖氨酸或细胞和组织粘合剂（见材料表）而得到提高。 </li><li>当细胞已经形成了融合层，他们已经准备好细胞间隙连接通讯检查。 </li></ol> 2，电穿孔程序电穿孔的细胞间隙连接通讯检查可以在层流罩外进行，因为只需要几分钟的时间。如果潜伏期较长时间都需要一个特定的实验，那么就可以完全在层流罩进行。在所有情况下，当细胞生长腔室必须是无菌的。 <ol><li>制备5毫克/毫升荧光黄液:将10毫克LY粉末（设置有电穿孔仪）在2ml无钙生长培养基中。对于实验中，需要较长的培育时间，过滤消毒溶液中，并储存在4℃。 </li><li>吸出培养基，用无钙培养基洗细胞，小心不要划伤单层或干细胞。如果细胞是干他们通常有较深的细胞核，并拿起LY没有电。的效果是在它更多地暴露于空气气流（ 图4F）滑动的中间更加明显。 </li><li>用Eppendorf移液器，吸管的染料溶液到细胞（400μl的整个腔室），在该腔室，b的边缘eing小心不要触摸细胞层。 </li><li>放置室与电穿孔仪附带的支架，并应用适当的强度（见讨论）的脉冲。 </li><li>小心吸一些LY解​​决方案。它可以重复使用的第二时间不太重要的实验。 </li><li>加入含10％透析血清钙的DMEM。 </li><li>孵育的细胞为3-5分钟，在培养箱中，以允许通过间隙连接的染料转移。 注:在这一点上列入透析血清帮助毛孔关闭。 </li><li>与钙的DMEM活细胞观察洗未掺入的染料。备选地，细胞可以固定在该阶段，通过加入4％甲醛的井，然后用PBS（磷酸盐缓冲盐水）。在所有情况下，清洗必须移除所有背景。 注:在初步实验，以确定最佳的条件下，如果电压过低，则有可能让细胞恢复在incubator表示了几分钟，电穿孔的同一张幻灯片第二次来保存幻灯片的成本。 </li></ol> 3，显微镜检查<ol><li>观察细胞的荧光下照射，使用倒置显微镜配备有适当的过滤器的染料被使用。对于荧光黄，激励是423nm，发射555nm处，全身振动过滤器尼康IX70显微镜。 </li><li>消除液面效应通过放置一个玻璃盖玻片上的塑料井的顶部，它填充到液体上方后，检查下相衬细胞形态。为更好的画面，以及可从滑动被分离，并在盖玻片放置在框架上。对于固定的细胞中，以及可填充有甘油的显微镜观察，而对于活细胞就必须充满生长培养基中。 注意:洗涤和拍摄是比较容易，如果下列染料（上述步骤2.7）的转移将细胞用甲醛固定。如果细胞是无吨固定，荧光从细胞取决于LY的介绍，而在固定的细胞的荧光被保持几个小时的细胞类型，电压和量消除内约60分钟。然而，荧光消失或O 2 / N孵化后消散，即便是在固定的细胞。出于这个原因，照片必须尽快电穿孔（ 图2）后采取。 </li></ol> 4，定量间通讯<ol><li>拍摄的细胞在荧光下，相位相反（见图3）20倍的目标。 </li><li>识别并用星标记电 ​​穿孔的细胞在边界与非电穿孔的区域（ 图2中 ，箭头，电穿孔边缘）。 </li><li>识别并以点标记的荧光细胞上的非电穿孔的一侧，其中所述染料已通过间隙连接的传输（ 图3A和3B）。 </li><li>除以总数通过电穿孔的细胞沿着边缘（ 图3A和3B）的数量的荧光细胞上的非电穿孔区域。 请注意:从至少200个连续的电穿孔的边缘细胞的转移计算每个实验。得到的数字是GJIC值。 </li></ol>

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The corresponding author is the inventor in a patent held by Queen&#8217;s University, which has been licensed to Cell Projects Inc. The company or the University had no influence upon the contents of the paper.

在协议中的关键步骤 电穿孔材料 该材料的纯度被电穿孔是非常重要的。如果细胞是非常平坦的，则高浓度的示踪染料必须使用（不超过20毫克/毫升为LY），并在这种情况下，纯度，甚至比在细胞有更多的球状1更为重要。除LY大量的各种其它染料或nonpermeant分子已被采用，例如为一系列的Alexa染料作为探针的通道由不同的连接蛋白25的使用。然而?...

电电流的一个小区中的应用程序会导致孔的细胞膜上形成，由一个过程被称为电穿孔。的孔允许的各种透过膜nonpermeant分子的通道。电场可以精确地控制，从而使形成的孔是非常小的和重新闭合迅速，以最小的干扰对细胞生理学。有趣的是，贴壁细胞可生长在涂有导电性和透明的铟锡氧化物（ITO）和电穿孔的原位载玻片上，即在那里生长的表面上，而不被分离的悬浮液中进行电穿孔。细胞可以生长得非常好在该表面上，并且当它们被安装并伸展，详细显微镜观察是可能的。使用这种技术，小型nonpermeant分子可即刻导入和成基本上100％的细胞，这使得该技术特别适用于研究排版的活化通路的继受体（1审查）的配体刺激onents。 电穿孔已经大多用于引入的DNA（也称为electrotransfection）。然而，电穿孔法在原位可用于引入了大量的各种分子，如肽2-4，寡核苷酸，如反义RNA，双链DNA诱饵寡核苷酸的宝贵抑制转录因子的结合，或siRNA 3,5， 6，放射性7-10个核苷酸，蛋白质11 12或前体药物13。在电穿孔之后，将细胞可被裂解为生化分析或固定和染色的抗体。 导电性ITO涂层非常薄，800-1,000Å，使细胞的生长不是由在两个表面的高度差的干扰，如在单元两端的导电涂层的边缘生长。这提供的优点是，非electroporated细胞可以生长并排电穿孔者，作为对照。同样的方法可以用于间隙连接，细胞间通讯（GJIC）的检查，如在视频中描述。 间隙连接是连接相邻的电池14的内部通道。间隙连接，细胞间通讯在肿瘤形成和转移中起重要作用，而癌基因如Src蛋白抑制GJIC 15,16。审查GJIC荧光染料如荧光黄（LY）经常被导入培养的细胞中，通过显微注射或刮除装17和染料扩散到邻近细胞下的荧光照明显微观察。这些技术不过总是导致细胞损伤。我们现在描述那里的细胞生长在其上部分涂有ITO 18载玻片的技术。电脉冲在LY（或其它染料）的存在下施加连载ca利用其渗透到细胞上滑动的导电部分越来越大，而染料迁移到相邻的，非电穿孔的细胞通过荧光照明显微观察。为了避免干扰敏感的细胞可能离开单层分离，被设计的组件，其并不需要一个电极被放置在小区的顶部施加的电流19。这种方法提供了定量GJIC在大量细胞的能力，而没有任何可检测的干扰细胞代谢，由下列血清刺激12没有在G1期的长度的影响如所示，增加在fos基因的水平原癌基因蛋白（Raptis，未发表）或两个与细胞应激时，p38的生猪或JNK / SAPK 激酶相关激酶。这种方法成为可能的癌基因的表达，转化和GJ的水平之间的关系的检查集成电路18，以及Src和Stat3的的后GJIC在多种细胞类型中，包括细胞肺肿瘤样本20-23新鲜培养的效果。此外， 原位电穿孔与所描述的设置，其中缺乏的顶电极已被成功地使用，用于在脂肪细胞分化的间隙连接闭合的示范，虽然细胞附着到基底减小在该阶段19,24。

<table><tbody><tr><td>DMEM</td><td>Cellgro http://www.cellgro.com/</td><td>50-013-PB</td><td></td></tr><tr><td>DMEM without Calcium</td><td>Hyclone (Thermo scientific: http://www.thermoscientific.com)</td><td>SH30319-01</td><td></td></tr><tr><td>Donor Calf Serum&amp;nbsp;</td><td>PAA: http://www.paa.com/</td><td>Cat.# B15-008</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>PAA: http://www.paa.com/</td><td>Cat.# A15-751</td><td></td></tr><tr><td>Electroporation apparatus</td><td>Cell Projects Ltd UK: http://www.cellprojects.com/</td><td>ACE-100</td><td></td></tr><tr><td>Chambers</td><td>Cell Projects Ltd UK</td><td>ACE-04-CC</td><td>4-wells</td></tr><tr><td>Chambers</td><td>Cell Projects Ltd UK</td><td>ACE-08-CC</td><td>8-wells</td></tr><tr><td>Lucifer Yellow</td><td>Cell Projects Ltd UK</td><td>ACE-25-LY</td><td>High purity</td></tr><tr><td>CelTak</td><td>BD Biosciences</td><td>354240</td><td>Cell and tissue adhesive</td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F1141</td><td></td></tr><tr><td>Collagen</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>Poly-Lysine</td><td>Sigma Aldrich</td><td>P8920</td><td></td></tr></tbody></table>

a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

图2显示了大鼠肝脏上皮T51B细胞22，在荧光下（图A和B），电穿孔和LY和拍照，或相差（C）的照明，以下定影和水洗。在图A中，将电穿孔的区域的边缘被标记为红色。荧光的红色线右边的梯度表示通过间隙连接的传输。在图3A和图3B中 ，间隙连接通讯的定量表示:将细胞在电穿孔的区域的边缘被确定，并用星号标记，而其中的染料转移了细胞被标记为一...

Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

a-functional-assay-for-gap-junctional-examination-electroporation

Research

JoVE Journal

Biology

9.9K 次观看.  Queen's University. This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

视频: A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

科研

癌症研究

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

粘附频率含量为原位跨交界的分子相互作用，在细胞与细胞界面的动力学分析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

生物工程

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

发育生物学

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

一个的趋电含量为神经前体细胞迁移的动力学分析外部施加直流电场

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

神经科学

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

生物学

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

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.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

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 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

一种碘-黄色荧光荧光蛋白-缝隙--细胞间通信方法

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...