Cell的传播分析单细胞显微注射
Summary
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。
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Lucifer yellow | Sigma | L0259 | |
| Lithium Chloride | Sigma | L4408 | |
| PBS tablets | Sigma | P4417 | |
| RPMI | Sigma | R4130 | |
| Bovine fetal serum | Cultilab | ||
| Trypsin | Sigma | T4799 | |
| Microscope | Nikon | TE-2000 | For microinjection experiments, one needs an inverted fluorescence microscope and filters for fluorescent microscopy |
| vibration-insulated table | Newport | VH3036W-OPT | A vibration-insulated table is needed to protect the experiments from vibration and avoid cell damage |
| Micromanipulator | Narishige | MMO-203 | This equipment allows precision adjustments of the micropipette, which is needed for cell micro injection. |
| Current Generator | Digitimer | DS2 | 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. |
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