对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电
Summary
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.
Abstract
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. 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 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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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’s University.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| DMEM | Cellgro http://www.cellgro.com/ | 50-013-PB | |
| DMEM without Calcium | Hyclone (Thermo scientific: http://www.thermoscientific.com) | SH30319-01 | |
| Donor Calf Serum | PAA: http://www.paa.com/ | Cat.# B15-008 | |
| Fetal Bovine Serum | PAA: http://www.paa.com/ | Cat.# A15-751 | |
| Electroporation apparatus | Cell Projects Ltd UK: http://www.cellprojects.com/ | ACE-100 | |
| Chambers | Cell Projects Ltd UK | ACE-04-CC | 4-wells |
| Chambers | Cell Projects Ltd UK | ACE-08-CC | 8-wells |
| Lucifer Yellow | Cell Projects Ltd UK | ACE-25-LY | High purity |
| CelTak | BD Biosciences | 354240 | Cell and tissue adhesive |
| Fibronectin | Sigma Aldrich | F1141 | |
| Collagen | BD Biosciences | 354236 | |
| Poly-Lysine | Sigma Aldrich | P8920 |
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